Functionalized cellulose nanospheres (spherical nanocellulose formates, SCNFs) were extracted by one-step HCOOH/HCl hydrolysis of lyocell fibers with cellulose II crystal structure. The as-prepared cellulose nanoparticles were characterized by field emission scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectrometry, X-ray photoelectron spectroscopy, and thermal gravimetric analysis. The influences of fiber-to-acid ratio on the yield, microstructure and properties of the SCNFs were studied. The SCNFs showed spherical nanoparticles with a narrow size distribution of 19–29 nm. X-ray photoelectron spectroscopy verified that carboxyl groups of HCOOH were firstly grafted on the primary hydroxyl groups of SCNFs and then secondary hydroxyl groups. Moreover, the maximum degradation temperature (Tmax) of SCNFs is as high as 365.4 °C due to preserving integrity of cellulose crystal structure, increased crystallinity and surface hydrophobic formate groups. This can enable these nanospheres as effective templates for the synthesis of nanohybrids or reinforcing material for high performance nanocomposites.