Owing to their low cost, high catalytic efficiency and biocompatibility, carbon-based metalfree catalysts (C-MFCs) have attracted intense interest for various applications, ranging from energy through environmental to biomedical technologies. While considerable efforts and progress have been made in mechanistic understanding of C-MFCs for non-biomedical applications, their catalytic mechanism for therapeutic effects has rarely been investigated. In this study, defect-rich graphene quantum dots (GQDs) were developed as C-MFCs for efficient ROS generation, specifically in the H2O2 -rich tumor microenvironment to cause multi-level damage of subcellular components (even in nuclei). While a desirable anti-cancer performance was achieved, the catalytic performance was found to strongly depend on the defect density. It is for the first time that the defect-induced catalytic generation of ROS by C-MFCs in the tumor microenvironment was demonstrated and the associated catalytic mechanism was elucidated. This work opens a new avenue for the development of safe and efficient catalytic nanomedicine.