A key aspect of biochip and biosensor preparation is optimization of the optical or electrochemical techniques that combine high sensitivity and specificity. Among them, optical techniques such as the use of fluorescent polymeric nanoparticles have resulted in dramatic progress in the field of diagnostics due to their range of advantages. We herein report a facile approach for the development of novel fluorescein polymeric nanoparticles (FPNPs) with immobilization of specific biomolecules for application in a highly sensitive optical biosensor. A series of three amphiphilic fluorescein polymers (poly(FMA--NAS--MA)), comprising hydrophobic fluorescein -methacrylate (FMA), hydrophilic -acryloxysuccinimide (NAS), and methacrylic acid (MA) monomers were synthesized through radical polymerization. In an aqueous environment, these fluorescein polymers self-assembled into spherical shaped nanoparticles with a well-defined particle size, narrow particle size distribution, and enhanced fluorescence properties. The bio-immobilization properties of the FPNPs were also tunable by control of the activated -hydroxysuccinimide ester group in the polymer series. Furthermore, the fluorescence sensitivity of bovine serum albumin detection by the FPNPs indicates that the limit of detection and sensitivity were improved compared to conventional fluorescence dye-labelled proteins. These novel FPNPs therefore represent a suitable technology for disease diagnosis and biomarker detection to ultimately improve the sensitivity of existing analytical methodologies in a facile and cost-effective manner.