Abstract: In order to improve the degradability and biocompatibility of sodium alginate (SA), SA was oxidized with sodium periodate and then grafted with cell adhesion peptide by carbodiimide water method. Modified sodium alginate (ROSA) was used as the material to prepare monodispersible water-in-oil droplets by the shear action of continuous relatively dispersed phase in microfluidic technology. After the release of calcium ions in the droplets, the droplets were crosslinked into homogeneous hydrogel microspheres. The structural of SA before and after modification were detected by infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The surface morphology and biocompatibility of the microspheres were characterized by inverted optical microscopy and laser confocal microscopy. The most suitable hydrogel microspheres for subsequent biomedical applications were obtained by systematically studying the effect of the fabrication technique on the microsphere properties, and adjusting the flow rates of the dispersed and continuum phases, the degree of oxidation and the mass fraction of the SA. Results showed that the formation of aldehyde group and the detection of nitrogen in ROSA proved the success of the SA material modification. The size of the hydrogel microspheres could be controlled at (50.4 ± 1.3) μm, the coefficient of variation was less than 5%, the monodispersion was good, and the structure shape was regular. The microspheres were completely degraded within 4 weeks in vitro and could effectively control the release of the loaded recombinant human bone morphogenetic protein-2 (rhBMP-2). The microspheres showed no obvious cytotoxicity to rat bone marrow mesenchymal stem cells (rBMSCs), and their adhesion to rBMSCs was significantly enhanced, showing good biocompatibility.