Crosslinked poly(vinyl alcohol) (PVA)/cellulose nanofibril (CNF) aerogel microspheres with two different particle sizes were prepared via a combination of the water-in-oil (W/O) emulsification process and the freeze-drying process. The oil phase and the cooling agent were optimized to ensure the frozen ice microspheres can be easily separated from the emulsion system. The aerogel microspheres are highly porous with bulk density as low as 0.0047 g/cm3 for the large microspheres. The pore size of the cellulose aerogel microspheres ranges from nano- to micrometers. Preliminary biocompatibility assay of the aerogel microspheres was investigated with NIH 3T3 cells to explore their potential application as scaffolds in tissue engineering. The well crosslinked aerogel microspheres were very robust that maintained their shape during the cell culture process. The live/dead assay of the cells showed that the cells could be seeded on the surface of PVA/CNF aerogel microspheres and accumulated successfully. It also demonstrated that microspheres fabricated in this work were nontoxic and biocompatible. The fluorescence images showed some of the cells migrated into the pores of the microspheres. Moreover, the large microspheres with bigger average pore size had more cell count than that of the smaller ones. This study confirmed the interconnected high porous nanofibrous structure of the PVA/CNF aerogel microspheres could facilitate the cell attachment, penetration and proliferation.