Abstract Background Microalgae are currently considered as a promising feedstock for the production of biofuels and high-value products. However, the efficient harvest of microalgal biomasses from their culture broth is a major challenge. The harvesting of algal biomass by flocculation combined with gravity sedimentation is more convenient and cost-effective than traditional methods such as centrifugation and filtration. Compared to inorganic and chemically synthetic flocculants, bioflocculants are a suitable choice for microalgal harvest due to their biodegradable and nontoxic properties. Nonetheless, the high production costs associated with expensive substrates hinder the commercial applications of bioflocculants. Previous studies have shown that the hydrolysates of lignocellulosic biomasses from dilute acid hydrolysis can be utilized as an inexpensive carbon source for the production of bioflocculants. However, the toxic by-products generated in the dilute acid hydrolysis step limit the efficiency of subsequent fermentation. The strains that produce bioflocculants by using untreated lignocellulosic materials can circumvent the pretreatment process, as well as promote the application of bioflocculants in microalgal harvest. Results Under alkaline fermentation conditions, the alkaliphilic strain Bacillus agaradhaerens C9 secreted 1.69 IU/mL of alkali-tolerant xylanase and 0.06 IU/mL of cellulase, indicating that this particular strain can efficiently convert untreated rice bran into bioflocculant (RBBF-C9), thereby circumventing rice bran pretreatment for downstream fermentation. The optimal fermentation conditions that result in the highest bioflocculant yield (12.94 g/L) were as follows: 20 g/L of untreated rice bran, 3 g/L of yeast extract, and 20 g/L of Na2CO3 at 37 °C for 24 h. RBBF-C9 contained 74.12% polysaccharides and 4.51% proteins, and was estimated to be 137 kDa. Furthermore, the bioflocculant RBBF-C9 exhibited good flocculating efficiency (91.05%) of oil alga Chlorella minutissima UTEX2341 when 60 mg/L of RBBF-C9 was added into the algal culture broth. Conclusions This study demonstrated that untreated rice bran is a suitable inexpensive substrate for the production of bioflocculants, and thus provides a novel approach in utilizing rice bran. The extracted bioflocculants may be potentially used in biomass harvesting of the oil algae C. minutissima UTEX2341 from the culture broth.