Bio-CaCO3 nanoparticles have several applications and have attracted significant attention in current research. N,N-dimethylformamide (DMF) has been proven to be an effective non-volatile solvent for synthesizing bio-CaCO3 nanomaterials from eggshell. However, the optimum ratio of eggshell and DMF need to be specified to achieve maximum nano-CaCO3 production for large-scale purposes. Thus, this work investigated the effect of eggshell/DMF mixing ratios on the production of CaCO3 nanoparticles from the chicken eggshell. The nano-CaCO3 were synthesized via dry milling and then sonication at a frequency of 40 kHz for 6 h in the presence of DMF. The eggshell mass was varied from 0.5 to 20 g per 100 mL of DMF. The synthesized CaCO3 materials were characterized using SEM, TEM, EDX, XRD, and BET surface analysis. The eggshell/DMF ratio was optimized to maximize the production of CaCO3 nanoparticles, and its effect on the size, crystallinity, surface area, and porosity of the CaCO3 particles were discussed. Increasing eggshell/DMF ratio decreased the sonication efficiency with increasing crystallite and particle size. The specific surface area of the synthesized CaCO3 particles decreased with increasing eggshell/DMF ratio. 1 g/100 mL was the optimum or highest ratio to obtain 100% nano-CaCO3. At 1 g/100mL ratio, the bio-CaCO3 contained a crystallite size of 23.08 nm, particle size between 5 and 30 nm and surface area of 47.44 m2 g−1.