Currently, oil spill pollution is one of the major environmental concerns for sea cucumber (Apostichopus japonicus) aquaculture. During oil spills, spraying chemical dispersants is generally considered an efficient oil spill response. However, the impact of chemical dispersant deployment during oil spills on sea cucumbers is still less known. In this study, we treated sea cucumbers with physically and chemically (by GM-2 chemical dispersant) dispersed Oman crude oil for 24 h. For antioxidant defense capacities, our results showed that physically dispersed crude oil caused a significant elevation on superoxide dismutase (SOD) and catalase (CAT) activities, and glutathione (GSH) content, while chemically dispersed crude oil caused a significant decrease in SOD activity and GSH content with no apparent change in CAT activity. As for non-specific immune responses, our results indicated that physically dispersed crude oil up-regulated acid phosphatase (ACP) and lysozyme (LZM) activities but had no obvious impact on alkaline phosphatase (ALP) activity. Differently, chemically dispersed crude oil down-regulated ACP and LZM activities while up-regulating ALP activity. Based on the integrated biomarker response analysis, the overall impact of chemically dispersed crude oil on antioxidant defense capacities and non-specific immune responses of sea cucumbers was more severe than physically dispersed crude oil.