The extensive therapeutic potential of protein drugs has been severely limited by their instability and short biological half-lives in vivo. To prolong their therapeutic effects, a sustained delivery system is required. In this study, cyclodextrin-based polymeric nanoparticles (CD-NPs), mineralized by calcium phosphate as the diffusion barrier, were developed as a carrier for sustained protein delivery. Spherical CD-NPs were readily prepared by a conjugate, composed of β-CD as the protein-binding moiety and carboxymethyl dextran as the substrate for mineralization in a physiological solution. Owing to the presence of carboxylic acids in CD-NPs, they were effectively mineralized by sequential addition of calcium nitrate and ammonium phosphate. The physicochemical characteristics of mineralized CD-NPs were characterized using FT-IR, thermogravimetric analysis, transmission electron microscopy and energy dispersive X-ray photoelectron spectroscopy. Mineralization reduced CD-NP particle size from 310 nm to 121 nm in PBS (pH 7.4) indicating the formation of compact nanoparticles. Carbonic anhydrase B (CAB), chosen as the model protein, was loaded into the mineralized CD-NPs with a high loading efficiency (80%) by a simple dialysis method. In vitro release tests showed that CAB was completely released from bare CD-NPs in 3 days. Interestingly, the mineralized CD-NPs released CAB in a sustained manner for 21 days, which was due to the stable calcium phosphate barrier inhibiting CAB release. The enzymatic activity of CAB, which was released from the nanoparticles, did not significantly deteriorate compared to native CAB. Overall, mineralized CD-NPs could be a promising carrier for sustained protein delivery.