[Objective] To establish a rapid, simple and efficient Agrobacterium-mediated genetic transformation system for Chlamydomonas reinhardtiii, we used the model organism C. reinhardtiii as the receptor material and optimized the Agrobacterium-mediated transformation system of C. reinhardtiii from two aspects: transformation method and transformants identification method. [Methods] We compared the effect of solid co-culture and liquid co-culture on the transformation efficiency of C. reinhardtii CC425 mediated by A. tumefaciens LBA4404. Besides, we analyzed the optimal reaction conditions and amplification efficiency of (1) two-step PCR after TE cleavage, and (2) one-step PCR without TE cleavage. [Results] The highest transformation efficiency was achieved by a 5-day liquid-medium co-culture of Agrobacterium and Chlamydomonas. The transformation rate was 43.33±1.67 transformants/10⁶ algal cells. The optimal reaction conditions were: amplification with high fidelity DNA polymerase Taq 1; the cell density involved in PCR was 5×10³–5×10⁶ cells/mL; before amplification, cells were boiled in TE lysis buffer for 20 min (two-step PCR method), or initial denaturation for 15 min (one-step direct PCR method). The amplification efficiency of two-step PCR is better than that of one-step PCR, but the latter is more concise. [Conclusion] Agrobacterium-mediated transformation system of C. reinhardtii was established and optimized, through which rapid genetic transformation can be fulfilled and the workload could be reduced.