FeO nanoparticles occupy a pivotal position in the realm of nanobiology due to their nontoxic, biocompatible, and superparamagnetic properties. This study examines the influence of surface modifiers on the properties of magnetic nanoparticles. Poly(methacrylic acid) (PMAA), poly(4-styrenesulfonic acid--maleic acid) sodium salt (PSSM), trisodium citrate (TSC), carboxymethylcellulose (CMC), and carboxymethylated-dextran 40 (CMD40) were introduced into a one-pot solvothermal method to synthesize magnetic nanoparticles. TEM, the 4-(bromomethyl)-6,7-dimethoxy coumarin (BMMC) absorption assay, and the Bradford method were employed to characterize the diameter, carboxyl content, and protein immobilization ability of the nanoparticles, respectively. The findings revealed that CMD40-modified magnetic nanoparticles (CMD40-MNPs) exhibited the highest carboxyl content and streptavidin (SA) immobilization content, reaching 6.5 × 10 mol/mg and 375 μg/mg, respectively. In contrast, CMC-modified magnetic nanoparticles displayed opposite trends. This is primarily attributed to dextran's unique molecular structure, which enhances its water solubility and biocompatibility, thereby facilitating contact with FeO nanoparticles in aqueous solutions. CMD40-MNPs possess a saturation magnetization value of 60.90 emu/g and can be collected within (60 ± 5) s using a standard magnetic separator. Cytotoxicity assays demonstrated that CMD40-MNPs are nontoxic to cells. A cell sorting strategy utilizing the binding of SA-CMD40-MNPs and biotin antihuman CD3 antibody-modified cell suspensions was employed to isolate CD3+T cells. The results indicate that the purity and efficiency of targeted CD3+T cells are 85.2% and 61.5%, respectively.