Abstract: To quantitatively evaluate the chemotaxis of the second-stage juveniles (J2s) of Meloidogyne incongnita, an experimental method using stereomicroscope camera and ImageJ grayscale statistics method to detect the chemotaxis of J2s by analyzing the movement trajectory of the root-knot nematode in Pluronic F-127 gel was established, and verified the reliability of the method with 10 reported compounds to induce chemotaxis. In addition, the method was used to determine the chemotaxis of 40 compounds against M. incongnita. The results showed that 22 compounds had attractive activity and 11 compounds had repellent activity at the concentration of 10 μg/mL, with p-anisidine and phloroglucinol being the most attractive and repellent to nematodes, respectively. Further studies showed that p-anisidine exhibited attraction activity at low concentrations and repellent activity against J2s of M. incongnita at high concentrations, while the repellent activity of phloroglucinol increased with higher concentrations. This study provides a reliable method for rapid and quantitative evaluation of the chemotaxis of compounds against M. incongnita and for the discovery of highly active root-knot nematode repellents.