Background: The purpose of our study is to develop the arthroscopic autofluorescence imaging (AFI) system to improve the visualization during arthroscopic surgery by real-time enhancing the contrast between joint structures with autofluorescence imaging. Its validity was evaluated around the arthroscopic anterior cruciate ligament (ACL) reconstruction, specifically improving the contrast between the femoral insertion site and its background. The feasibility of the AFI system was validated with bovine and human knees. The spectral responses of the femoral insertion site and its surrounding bone and cartilage were measured with a fluorospectrometer. A prototype of the AFI system was developed based on the spectral responses (SR) and test images of the insertion site. The accuracy was validated by evaluating the overlap between manually segmented insertion sites on the white light color images and on the corresponding spectral unmixed autofluorescence images. The final prototype of the AFI system was tested during arthroscopy in cadaveric knees. Results: The results showed that the joint structures have different SRs. Spectral unmixing enabled separation of the SRs and improved the contrast between the joint structures. The agreement between visible light and autofluorescence ligament insertions had a mean Dice coefficient of 0.84 and the mean Dice coefficient of the interobserver variability for visible light imaging was 0.85. Conclusions: We have shown that the femoral insertion site can be accurately visualized with autofluorescence imaging combined with spectral unmixing. The AFI system demonstrates the feasibility of real-time and subject-specific visualization of the femoral insertion site which can facilitate anatomic ACL reconstruction. In addition, the AFI system can facilitate arthroscopic procedures in other joints and can also be used as a diagnostic tool.