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The purpose of the present study is to evaluate whether ethanol concentrations (20-95% v/v), extraction time (30-240 min) and extraction temperature (20-80℃) will affect the antioxidative polyphenols extraction utilising one variable at a time (OVAT) method. Total phenolic content (TPC), total flavonoid content (TFC) were used to determine the polyphenolic amount while 2,2-Diphenyl -1-picrylhydrazyl (DPPH) radical scavenging activity was used for evaluating the primary antioxidant capacity of red pitaya defatted seed (RPDS). All various extracting conditions studied showed a significant effect at p < 0.05 on the amount of phenolic and antioxidant activity. The optimise conditions determined for extracting phenolic RPDS were 80% ethanol for 60 min at 80 ℃, which correspond to the value of 104.76 mg GAE/g sample for TPC, 10.76 mg QE/g sample for TFC with 89.36% inhibition of DPPH radical. This study suggests that RPDS extract have the potential to be utilised as halal and natural antioxidants agent in the future product formulation.