In this study, we developed a simple method for preparing highly dispersed, stable, and streptavidin (SA)-functionalized carboxymethyl dextran (CMD)-coated melamine nanoparticles (MNPs) in an aqueous buffer at neutral pH. Dynamic light scattering (DLS) revealed the agglomeration of MNPs in an aqueous buffer at neutral pH. When CMD, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were simultaneously mixed with the MNPs, CMD was bound to the MNPs, promoting their dispersibility. Preparation of SA-CMD-MNPs was accomplished simply by adding SA solution to the CMD-MNPs. The amount of SA bound to the CMD-MNPs was quantified by the bicinchoninic assay, and the amount of SA molecules bound to each CMD-MNP was 417 ± 4. SA-CMD-MNPs exhibited high dispersity (polydispersity index = 0.058) in a neutral phosphate buffer and maintained it for 182 days with dispersion using a probe sonicator (5 s) before DLS characterization. The performance of the SA-CMD-MNPs in biosensing was evaluated by immunohistochemistry, which revealed that the nanoparticles could specifically stain MCF-7 cells derived from breast cancer cells with low HER2 expression. This study provides an effective method for synthesizing highly dispersible nanoparticles for biosensing.