In this study, changes in the molecular mass of alginate were investigated during its enzymatic degradation and the processing parameters for the enzymatic preparation of alginate oligosaccharides were explored. Furthermore, the in vitro immunological activity of degraded products with different molecular mass was evaluated. The results showed that the molecular mass of alginate decreased significantly after degradation by alginate lyase, and three degradation products with different molecular mass were obtained through gradient ethanol fractionation. Their weight-average molecular masses were 13.4, 5.73 and 3.85 kDa, respectively. Using single factor experiments, the optimal processing parameters were determined as pH 7.0, alginate lyase dosage 15 U/g substrate, and hydrolysis time 24 h, giving a yield of 28.05%. All alginate and its degraded products had immunoenhancing activity in mouse macrophages, and among them, the effect of the product with a weight-average molecular mass of 5.73 kDa was most pronounced and more pronounced than that of alginate oligosaccharides. By adding TAK-242, a blocker of macrophage Toll-like receptor 4 (TLR4), it was verified that the degraded products of alginate regulated macrophage immune activity by inducing the secretion of TLR4 and consequently causing cascade reactions to increase the secretion of NO, TNF-α and IL-6. These results can provide a theoretical basis for the high-value utilization of alginate.