Updated project metadata. We herein report a photo-oxidation driven proximity labeling strategy to profile mitochondrial proteome by light dependence in living cells with high spatiotemporal resolution. Taking advantage of organelle-localizable organic photoactivated probe generating reactive species and nucleophilic substrate for proximal proteins oxidation and trapping, mitochondrial proteins were selectively labeled by spatially limited reactions in native environment. Integration of photo-oxidation driven proximity labeling and quantitative proteomics facilitated the plotting of mitochondrial proteome in which up to 310 mitochondrial proteins were identified with the specificity of 64% in HeLa cells. Furthermore, mitochondrial proteome dynamics was deciphered in drug resistant Huh7 and LPS stimulated HMC3 cells which were hard-to-transfect. A number of differential proteins were quantified which were intimately linked to critical processes and provided insights into the related molecular mechanisms of drug resistance and neuroinflammation in the perspective of mitochondria.