The effects of cellulose particle sizes of lime peel on biofilm were studied in this work. The cellulose particle sizes, i.e., 355, 250, 125, and 75 µm, were examined for biofilm preparation. The XRD patterns revealed a main phase of cellulose in all samples. The presence of secondary phase did not affect biofilm preparation. The FTIR spectra indicated that the decrease in cellulose particle size enhanced carboxyl and hydroxyl groups of carboxymethyl cellulose (CMC) powder. The cellulose particle size 75 µm had a strong presence of hydroxyl and carboxyl groups. This contribution significantly promoted high water solubility of CMC powder from 75 µm and high-quality biofilm with smooth surface and homogeneity. The optical band gap of biofilms was about 4.75-4.88 eV. With these results, the investigation here revealed that the lime cellulose particle size 75 µm produced high quality biofilm, which may be alternative bioproduct in the future.