Highly homogenous and spherical shaped Selenium nanoparticles with an average size of 20 nm were synthesized from an eco-friendly and environmentally safe approach using bovine milk protein casein as stabilizing agent (named as CasSeNPs). Protein functionalization provided excellent stability at variable temperatures (25–60 °C), pH (8–10), ionic strength (0.05–2 M), and storage period for more than five months. Synthesized CasSeNPs were extensively characterized using various spectroscopic (UV–visible, Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy), particle sizing (dynamic light scattering (DLS) and zeta potential), microscopic (scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM/EDS), atomic force microscopy (AFM)), and X-ray diffraction (XRD) measurements. Moreover, CasSeNPs was established as highly sensitive and selective sensor for the simultaneous detection of vanadium (V+2) and copper (Cu+2) ions with significant linearity (0.5–10 and 0.1–1.0 µM, respectively), detection limits (LOD = 0.03 and 0.067 µM, respectively), and percent recoveries (94–105 %) in real environmental (tap water) and biological (human plasma and urine) samples with adequate test performance. Cumulatively, obtained results revealed that the established very simple, biocompatible and economical nanosensor has great potential for applications in analytical chemistry as well as for biological investigations.