Anti-lipopolysaccharide factor is a class of antimicrobial peptides with lipopolysaccharide-binding structural domains, which has a broad antimicrobial spectrum, high antimicrobial activities, and broad application prospects in terms of the aquaculture industry. However, the low yield of natural antimicrobial peptides and their poor expression activity in bacteria and yeast have hindered their exploration and utilization. Therefore, in this study, the extracellular expression system ofChlamydomonas reinhardtii, by fusing the target gene with the signal peptide, was used to express anti-lipopolysaccharide factor 3 (ALFPm3) fromPenaeus monodonin order to obtain highly active ALFPm3. TransgenicC. reinhardtiiT-JiA2, T-JiA3, T-JiA5, and T-JiA6, were verified using DNA-PCR, RT-PCR, and immunoblot. Additionally, the IBP1-ALFPm3 fusion protein could be detected not only within the cells but also in the culture supernatant. Moreover, the extracellular secretion containing ALFPm3 was collected from algal cultures, and then its bacterial inhibitory activity was analyzed. The results showed that the extracts from T-JiA3 had an inhibition rate of 97% against four common aquaculture pathogenic bacteria, includingVibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus,andVibrio parahaemolyticus. The highest inhibition rate of 116.18% was observed in the test againstV. anguillarum. Finally, the minimum inhibition concentration (MIC) of the extracts from T-JiA3 toV. harveyi, V. anguillarum, V. alginolyticus,andV. parahaemolyticuswere 0.11 μg/μL, 0.088 μg/μL, 0.11 μg/μL, and 0.011 μg/μL, respectively. This study supports the foundation of the expression of highly active anti-lipopolysaccharide factors using the extracellular expression system inC. reinhardtii, providing new ideas for the expression of highly active antimicrobial peptides.