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Microalgae play an important role in the formation of biofloc. To demonstrate the feasibility of <i>Chlorella pyrenoidosa</i> in biofloc formation, an experiment was performed with a simple random design consisting of five inoculation levels (in triplicate) of <i>C. pyrenoidosa</i> (0, 1 × 10⁸, 1 × 10⁹, 5 × 10⁹, and 1 × 10¹⁰ cells·L⁻¹) in the biofloc system. All treatments kept a C:N ratio of approximately 15:1. This study observed the effects of different initial concentrations of <i>C. pyrenoidosa</i> on biofloc formation, water quality and bacterial community in biofloc systems. The results indicated that <i>C. pyrenoidosa</i> had the ability to enhance biofloc development, especially when the <i>C. pyrenoidosa</i> initial concentration reached 5~10 × 10⁹ cells·L⁻¹. Too high or too low a concentration of <i>C. pyrenoidosa</i> will adversely affect the formation of biofloc. The effect of <i>C. pyrenoidosa</i> addition on water quality (TAN, NO₂⁻-N, and NO₃⁻-N) was not significant in the final stage. The inoculation of <i>C. pyrenoidosa</i> decreased the species richness and diversity of the bacterial community but increased the domination of <i>Proteobacteria</i> and <i>Bacteroidota</i> in the biofloc system, especially the order of <i>Rhizobiales</i>. The addition of <i>C. pyrenoidosa</i> could maintain water quality by increasing the proportion of several denitrifying bacteria, including <i>Flavobacterium</i>, <i>Chryseobacterium</i>, <i>Pseudomonas</i>, <i>Brevundimonas</i>, <i>Xanthobacter</i>, etc. These above dominant denitrifying bacteria in the biofloc system could play a major role in reducing the concentration of NO₂⁻-N and NO₃⁻-N. So, we recommended the reasonable concentration is 5~10 × 10⁹ cells·L⁻¹ if <i>C. pyrenoidosa</i> is used to rapidly produce biofloc.