The decellularization of plant tissues can be one of the design options of scaffolds in tissue engineering. Chemical detergents such as Triton X‐100 and sodium dodecyl sulfate (SDS) in different concentrations were used to decellularize olive leaves as an acellular plant matrix for tissue engineering. The samples were investigated by different analyses such as Hematoxylin and Eosin staining, SEM, tensile strength, swelling, water vapor transmission, and toxicity. The results of staining and toxicity tests showed that the Triton X‐100 decellularized samples at a concentration of 0.1% had the best morphology and the lowest toxicity. Mechanical results showed that the elasticity modulus of acellular samples was significantly reduced compared to normal leaf samples. While swelling rate and water vapor transmission in acellular samples compared to the control sample doubled and tripled, respectively. In general, acellular olive leaf can be suggested as a scaffold for tissue engineering applications.