The aim of the present investigationwas plan to evaluate the antioxidant potential of Extracellular Polymeric substances (EPS)from the marine actinobacterium of Actinomadura sp.T3S5. Marine actinobacteria organisms are potentially known for antimicrobial, anticancer, antiviral, insecticidal and enzyme inhibitory activities. The extracts identified some of the major phenolic compounds, which might be responsible for the antioxidant potential and cyto-protection. This primary study was to examine whether EPS from Marine Actinobacterium of Actinomadura has potentially to show in vitro antioxidant property.The antioxidant potential of Extracellular polymeric substance was tested by DPPH, Total antioxidant assay and scavenging of nitric oxide assay. The antioxidant activity by DPPH method (48%), inhibition of total antioxidant activity(59%), and Nitric oxide (47%). Marine actinobacterium of Actinomadura is greatly suggested for beneficial biomedical applications and in the development of new eco-friendly biomaterial for environmental cleanup.