Chitosan oligosaccharide (COS) is a potent immunopotentiator, but rarely used in aquaculture. The aim of this study is to investigate the effect of feeding COS intermittently on growth, non-specific immune indices, digestive enzyme activities, histological and disease resistance of sea cucumber Apostichopus japonicus. A. japonicus [mean body weight (18.51±0.28) g] were first fed for 8 weeks by two groups (control group, A. japonicus were fed with basal diet; treatment group, sea cucumbers were first fed with basal diet added 0.5% COS for 1 day and then fed with basal diet for 2 days). Growth performance, non-specific immune enzyme activities, intestinal enzyme activities, the structure of the respiratory tree, intestines，expression of intestinal immune gene and immune response were measured to investigate the effects of feeding frequency with COS once every 3 days of sea cucumber A. japonicus. The results indicated that treatment group had significantly higher ratio of visceral weight to body wall weight (VBR) and ratio of intestine weight to body wall weight (IBR) (P<0.01). Phagocytic activity and respiratory outburst of coelomocytes in treatment group were significantly higher than those in control group (P<0.05). The activities of enzymes such as acid phosphatase (ACP), falkaline phosphatase (AKP), lysozyme (LZM) and total nitric oxide synthase (T-NOS) of intestine in treatment group increased significantly (P<0.01) and the activities of AKP and LZM of intestine in treatment group increased by 70.06% and 156%, respectively. The mRNA relative expression of Aj-lyz gene (P<0.01) of intestine in treatment group were significatly higher than that in control group, and the relative expression level of Aj-lyz gene of intestine in treatment group increased by 22.04%, respectively. The activity of catalase (CAT) of coelomocytes in treatment group was significantly higher than that in control group (P<0.01), but the content of malondialdehyde (MDA) of coelomocytes and intestine showed no significant difference between the two groups (P>0.05). Compared with the control group, the muscularis and serosa in foregut, fold height and fold width in midgut and hindgut were significantly increased in treatment group (P<0.01). We found that the survival rate of the sea cucumber A. japonicus against infection with Vibrio splendidus was higher in the treatment group, with a relative protection rate of 66.67%. Above all, the feeding frequency of once every 3 days for COS is recommended to promote the growth performance, non-specific immunity and immune response of the A. japonicus. This study provides theoretical support for follow-up research.