<p><i>Picochlorum maculatum</i> MACC3 is a marine picoalga rich in essential nutrients, including polyunsaturated fatty acids (PUFAs) and carotenoids. Preliminary research showed that chitosan-flocculated <i>P. maculatum</i> MACC3 enhanced the growth of <i>Penaeus monodon</i> post-larvae. This study aimed to identify the most effective biomass form for feed supplementation and evaluate the contributions of chitosan and the alga on the health of <i>P. monodon</i> post-larvae. A feeding trial was conducted for 60&#xa0;days with six experimental groups: T1 (10% freeze-dried alga), T2 (10% algal paste), T3 (10% chitosan-flocculated freeze-dried alga), T4 (10% chitosan-flocculated algal paste), T5 (0.4% chitosan), and C (control group fed with commercial feed). All treatment groups demonstrated significantly enhanced growth performance compared to the control group, with T4 exhibiting the most substantial improvements in growth and survival. The digestive enzyme activities (amylase, protease, and lipase) were markedly higher in the treatment groups, particularly in the T4 group (2–3 fold increase). Following the white spot syndrome virus (WSSV) challenge, the T4 group demonstrated the highest extended survival (40%) and significantly increased immune-related enzyme activities, including protease, catalase, and prophenol oxidase. Additionally, the expression of immune-related genes, including alpha-2-macroglobulin (<i>A2M</i>), crustin (<i>crustin</i>), prophenoloxidase (<i>proPO</i>), and transglutaminase (<i>TGase</i>), was significantly upregulated in the T4 group compared to the control. Notably, <i>TGase</i> expression showed a remarkable 6-fold increase, underscoring the enhanced immune response in the T4 group. The results demonstrated that combining <i>P. maculatum</i> MACC3 and chitosan, particularly in the paste form, provided significant health benefits for <i>P. monodon</i> post-larvae and can be integrated into shrimp feed formulations to support sustainable aquaculture.</p>