The position and configuration of the C═C bond have a significant impact on the spatial conformation of unsaturated lipids, which subsequently affects their biological functions. Double bond isomerization of lipids is an important mechanism of bacterial stress response, but its in-depth mechanistic study still lacks effective analytical tools. Here, we developed a visible-light-activated dual-pathway reaction system that enables simultaneous [2 + 2] cycloaddition and catalytic cis-trans isomerization of the C═C bond of unsaturated lipids via directly excited anthraquinone radicals. Density functional theory calculations revealed the oxygen radical addition transition state and the addition-elimination isomerization mechanism of the reaction. A full-dimensional resolution method for C═C bond position and configuration was developed based on the bifunctional reaction and liquid chromatography-mass spectrometry. This method was then applied to the study of bacterial environmental stress response mechanisms. The C═C bond cis-trans and positional isomerization patterns of Pseudomonas membrane lipids under temperature stress were discovered, and the effect of temperature stress on fatty acid biosynthesis was also revealed. This study not only provides an effective tool and key information for the study of bacterial stress response mechanisms, but also enriches the toolbox of visible light chemical reactions.