Ginkgo biloba is utilized as food, medicine, wood, and street trees among other things. The objective of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for gender distinction of G. biloba . Male-specific SCAR gene can be utilized to identify G. biloba gender using LAMP. The optimized LAMP conditions, temperature 60 °C, 2-mM MgSO 4 , and [F3/B3]:[FIP/BIP] primer ratio of 1:4 were selected as final conditions. The G. biloba SCAR LAMP displayed a sensitivity of 10 ng when amplified by concentration under the optimum conditions. Additionally, it demonstrated a particular response in male with SYBR Green I in LAMP analysis that can be a more powerful tool for field and scale-up applications. Our work represents a first attempt to identify G. biloba gender using LAMP and offers an efficient and reliable tool for roadside landscaping. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12033-023-00673-7.