Abstract: Biological nitrification inhibitors (BNIs) are natural bioactive substances released by plants through root exudation, tissue extraction, or litter decomposition, capable of selectively inhibiting ammonia-oxidizing microorganisms and the activity of their key enzymes, thereby slowing the conversion of ammonium nitrogen (NH₄⁺-N) to nitrate nitrogen (NO₃⁻-N). This process effectively reduces nitrogen leaching and N₂O emissions, thus enhancing nitrogen fertilizer use efficiency. Based on a bibliometric analysis of the literature, this study reveals that research interest in BNIs has increased rapidly since 2020, with research themes progressively shifting from fundamental mechanisms toward applied strategies. By synthesizing domestic and international studies published between 2000 and 2025, this review systematically summarizes the types and sources of BNIs and highlights that various crops, including Brachiaria, sorghum, wheat, rice, maize, and sugarcane, are capable of releasing active BNI compounds. Different BNIs exhibit substantial variability in inhibitory strength, environmental stability, and soil adaptability, and generally display pronounced pH dependency. Hydrophilic BNIs, such as methyl p-hydroxyphenyl propionate and 1,9-decanediol, are regulated by rhizosphere pH, nitrogen form, and plasma membrane H⁺-ATPase activity and are released across membranes via transporter-mediated pathways, whereas hydrophobic BNIs, such as sorgoleone, rely primarily on vesicle trafficking and exocytosis. In addition, external environmental factors, including soil moisture, bulk density, and oxygen availability, significantly influence BNI release. BNIs mainly inhibit nitrification by suppressing the activities of ammonia monooxygenase and hydroxylamine oxidoreductase, thereby blocking the first step of the nitrification process. Some BNIs may also interfere with electron transport chains or scavenge nitric oxide intermediates. Moreover, increasing attention has been paid to the regulatory effects of BNIs on nitrite oxidoreductase and key denitrification genes, such as nirK and nosZ, revealing their potential for coordinated regulation across multiple steps of the nitrification-denitrification pathway. Advanced analytical techniques, including chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, have been widely applied for the structural identification of BNIs, facilitating their accurate and efficient detection. Overall, BNIs demonstrate strong potential for the coordinated mitigation of NO₃⁻ leaching, NH₃ volatilization, and N₂O emissions in agricultural systems, contributing to both enhanced nitrogen use efficiency and environmental protection. However, their field stability and regional adaptability remain insufficiently understood. This review further summarizes current application methods and technologies of BNIs, focusing on three major aspects: breeding and improvement of BNI-producing crops, the establishment of crop rotation and intercropping systems involving BNI-producing species, and the development of green nitrogen fertilization technologies based on BNIs. Potential approaches to improve field application efficiency and environmental adaptability are discussed. In view of the existing challenges in agricultural application, this study analyzes key bottlenecks, including the variability of BNI effectiveness across different soil conditions, the lack of commercial products and large-scale field applications, and the absence of standardized application rates. The importance of establishing robust regulatory and policy frameworks to promote the industrialization and large-scale adoption of BNIs is emphasized. Future research should prioritize high-throughput BNI screening, elucidation of molecular regulatory mechanisms, multi-omics-based functional analyses, and synergistic integration with crop breeding, thereby facilitating stable application across diverse environments and the implementation of supporting policy systems.