Graphene exhibits exceptional electronic properties, superior mechanical strength, and remarkable flexibility, driving significant advances in flexible electronics. However, achieving high-precision patterned graphene via in situ fabrication for such applications remains challenging, limiting the development of graphene-based flexible devices. In this study, we successfully synthesized patterned graphene with high precision by substrate surface oxidation technology. The effect of substrate surface oxidation on patterned graphene growth was deeply investigated. By regulating the oxidation time, we precisely controlled the oxidation degree of the substrate and characterized the boundary precision between oxidized and unoxidized regions. Finally, we achieved the high-precision in situ fabrication of patterned graphene with a feature size of 0.5 μm on selectively oxidized substrates. Furthermore, we fabricated a flexible fluorescent device based on patterned graphene, demonstrating the pronounced fluorescence quenching effect of graphene (/ ≈ 3).