This study establishes an Agrobacterium-mediated transformation platform for the unicellular microalga Chlorella sp. (UTEX B 3198). The microalga was cultivated in BG11 and Modified Bold 3N media, with total biomass and relative growth rate analyzed as key metrics to optimize downstream processes. The Agrobacterium tumefaciens strain LBA4404, carrying the pCAMBIA1305.1 plasmid (Add Gene plasmid number: 64401), expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter genes under a 35S promoter, was utilized for transformation. Several critical factors were systematically explored to maximize transformation efficiency. These included IPTG concentration, selection antibiotics, the ratio of microalga to Agrobacterium, and medium ratios, all of which were carefully optimized to ensure successful integration of foreign genes. The approach provided a high degree of precision in balancing these parameters, thereby enhancing the overall efficiency and reproducibility of the transformation process. The use of both GFP and GUS as reporter genes offered dual validation techniques, enabling real-time visualization of gene expression and enzymatic assays for deeper functional analysis. This dual approach not only confirmed the presence of the introduced genes but also allowed for the assessment of their active expression in the microalga. Such versatility in validation methods strengthens the platform’s potential for broader applications in microalgal biotechnology. Molecular confirmation of successful transformation was achieved through PCR amplification of the GFP and GUS reporter genes, providing clear evidence of gene expression in Chlorella sp. (Figure 1 a-h). This molecular validation reinforced the robustness of the platform, offering a reliable and efficient method for genetic engineering in Chlorella, with potential applications in biofuel production, environmental monitoring, and other biotechnological fields.