The purpose of this study was to produce and evaluate different mechanical, physical and in vitro cell culture characteristics of poly(L-lactic) acid (PLLA) interference screws. This work will focus on evaluating the effect of two important parameters on operation of these screws, first the tunnel diameter which is one of the most important parameters during the operation and second the thermal behavior, the main effective characteristic in production process. In this work, PLLA screws were produced by a two-stage injection molding machine. For mechanical assessment of the produced screws, Polyurethane rigid foam was used as cancellous bone and polypropylene rope as synthetic graft to simulate bone and ligament in real situation. Different tunnel diameters including 7–10 mm were evaluated for fixation strength. When the tunnel diameter was changed from 10 to 9 mm, the pull-out force has increased to about 12 %, which is probably due to the aforementioned frictional forces, however, by reducing the tunnel diameter to 8 and 7 mm, the pull-out force reduced to 16 and 50 % for 8 and 7 mm tunnel diameter, respectively. The minimum and maximum pull-out force was obtained 160.57 and 506.86 N for 7 and 9 mm tunnel diameters, respectively. For physicochemical assay, Fourier transform infrared spectroscopy (FTIR), degradation test and differential scanning calorimetry (DSC) were carried out. The crystallinity (Xc) of samples were decreased considerably from 64.3 % before injection to 32.95 % after injection with two different crystallographic forms α′ and α. probably due to the fast cooling rate at room temperature. In addition, MTT and cell attachment assays were utilized by MG63 osteoblast cell line, to evaluate the cytotoxicity of the produced screws. The results revealed no cytotoxicity effect.