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Background: Gastrointestinal stromal tumor (GIST) is currently regarded as a potentially malignant tumor, and early diagnosis is the best way to improve its prognosis. Therefore, it will be meaningful to develop a new method for auxiliary diagnosis of this disease. Methods: Here we try out a new means to detect GIST by combining two-photon imaging with automatic image processing strategy. Results: Experimental results show that two-photon microscopy has the ability to label-freely identify the structural characteristics of GIST such as tumor cells, desmoplastic reaction, which are entirely different from those from gastric adenocarcinoma. Moreover, an image processing approach is used to extract eight collagen morphological features from tumor microenvironment and normal muscularis, and statistical analysis demonstrates that there are significant differences in three features—fiber area, density and cross-link density. The three morphological characteristics may be considered as optical imaging biomarkers to differentiate between normal and abnormal tissues. Conclusion: With continued improvement and refinement of this technology, we believe that two-photon microscopy will be an efficient surveillance tool for GIST and lead to better management of this disease. [ABSTRACT FROM AUTHOR]