In situ soil zymography can conveniently and intuitively display the two-dimensionally spatial variation of enzyme activity. Coupling with other imaging techniques and analysis methods, in situ soil zymography has been developed into a highly targeted method for analyzing soil micro-ecological processes and interface processes of soil-root-soil animal interaction. However, in situ soil zymography is immature. It is urgent to analyze its technical deficiencies and improve the technical methods. It is also necessary to hackle its application in different disciplines nowadays, and furtherly expand and deepen its application prospects. This paper summarizes and analyzes the application status of in situ soil zymography technology in the field of soil enzyme related research, combining with the optimization of substrate carrier selection and enzyme activity quantification of in situ soil zymography technology, analyzing its existent problems. The results show that:① the types of enzymes available for research are less; ② the accuracy of the results is still questionable; ③ the research in other fields except rhizosphere research is less and lack of systematism, so it is difficult to use for reference directly. These are still the biggest obstacles to the application of in situ zymography in many fields. In conclusion, although there are still some defects in in-situ soil zymography, it has great advantages of simple operation and high spatial and temporal resolution of measurement results. This paper provides theoretical guidance for promoting the application and development of soil in situ zymography in soil science and ecology.