A facile, eco-friendly fluorescence approach based on the biogenic formation of zinc oxide nanoparticles using the biomass of Plicosepalus curviflorus shoots was developed. The suggested approach was employed to analyze three phenolic compounds (catechin, curviflorside, and curviflorin) isolated from the shoots of P. curviflorus. The surface morphology of the prepared ZnONPs was characterized by carrying out different microscopic and spectroscopic investigations. A significant UV-Vis absorption peak of ZnONPs was recognized at 345 nm and the FT-IR spectra of the isolated catechin, curviflorside, and curviflorin in the presence of sodium dodecyl sulfate (SDS) and ZnONPs were recorded at λem 470, 490, and 484 nm after excitation at λex 380, 420, and 410 nm. The suggested fluorescence method displayed linear concentration ranges of 10–120, 5–100, and 10–150 μg mL−1 for the three isolated compounds, respectively. The shoot extract, isolated compounds, and ZnONPs were screened for antibacterial and anticancer effects against four different types of bacterial strains and HeLa cells, respectively. The ZnONPs exhibited the highest zone of inhibition against Escherichia coli and Staphylococcus aureus strains when compared with pure, isolated compounds and shoot extract. The anticancer potential of ZnONPs (64%) was stronger as compared to the 160 µg mL−1 of shoot extract (49%), catechin (52%), curviflorside (54%), and curviflorin (58%) at 160 µg mL−1. Moreover, all the samples were investigated for hemolysis activity and showed a potent anti-hemolytic effect. The developed analytical method showed excellent sensitivity and reliability for the concurrent analysis of the isolated bioactive markers.