Many approaches have been developed to regenerate biological substitutes for repairing damaged tissues. Guided bone/tissue regeneration (GBR/GTR) that employs a barrier membrane has received much attention in recent years. Regardless of substantial efforts for treatment of damaged tissue in recent years, an effective therapeutic strategy is still a challenge for tissue engineering researchers. The aim of the current study is to fabricate a GBR membrane consisting of polycaprolactone (PCL)/gelatin/chitosan which is modified with different percentages of beta-tricalcium phosphate (beta-TCP) for improved biocompatibility, mechanical properties, and antibacterial activity. The membranes are examined for their mechanical properties, surface roughness, hydrophilicity, biodegradability and biological response. The mechanical properties, wettability and roughness of the membranes are improved with increases in beta-TCP content. An increase in the elastic modulus of the substrates is obtained as the amount of beta-TCP increases to 5% (145-200 MPa). After 5 h, the number of attached cells is enhanced by 30%, 40% and 50% on membranes having 1%, 3% and 5% beta-TCP, respectively. The cell growth on a membrane with 3% of beta-TCP is also 50% and 20% higher than those without beta-TCP and 5% beta-TCP, respectively. Expression of type I collagen is increased with addition of beta-TCP by 3%, while there is no difference in ALP activity. The results indicated that a composite having (3%) beta-TCP has a potential application for guided bone tissue regeneration.