Diatomite was modified by chitosan to prepare modified diatomite, and the modified diatomite in an optimized ratio was utilized in coal bio-flocculation. The interaction behavior and flocculation mechanism of modified diatomite on coal slurry water were investigated by single factor experiments, infrared spectroscopy, BET measurements, and zeta potential measurements. The single factor experiments showed that when the amount of microbial flocculant added was 1.5 ml, the temperature of coal slurry water was 39 °C, the pH was 5, and the amount of modified diatomite was 0.2 g, after 30 min of sedimentation, the flocculation transmittance of the coal slurry water reached 84.3%. The infrared spectra showed that the -NH2 and -OH of the chitosan molecule had a polar interaction with the Si-OH bond in diatomite. The BET measurements showed that the specific surface area of diatomite was not a decisive factor affecting the flocculation effect. Zeta potential measurements indicated that the amino protonation of chitosan increased the isoelectric point (IEP) of modified diatomite. These results showed that modified diatomite has a good effect on coal bio-flocculation.