To elucidate the infection mechanism of Penicillium oxalicum strain FS-PNC isolated from the cadavers of infected brown planthoppers (Nilaparvata lugens)—against rice planthoppers, the green fluorescent protein (GFP) gene was introduced into FS-PNC via a polyethylene glycol (PEG)-mediated protoplast transformation method, successfully constructing the labeled strain FS-PNC-GFP. The mycelia, conidiophores, and conidia of the labeled strain stably expressed GFP, indicating successful integration and functional expression of the GFP gene in the fungal genome. Pathogenicity assays demonstrated that FS-PNC-GFP caused cumulative corrected mortality rates exceeding 70% for the third-instar nymphs and over 80% for the adults of three rice planthopper species: Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus. No significant differences in pathogenicity were observed among the three planthopper species, and the virulence of FS-PNC-GFP was comparable to that of the wild-type strain FS-PNC, indicating that GFP labeling did not affect its pathogenic activity. The GFP-tagged strain constructed in this study retains the high pathogenicity of the wild-type strain against rice planthoppers while offering excellent traceability, providing a key tool for further clarifying the infection pathways of FS-PNC and laying a foundation for optimizing its field application as a biocontrol agent and deeply exploring host-fungus interaction mechanisms.