Up-conversion luminescence (UCL) in crystalline rubrene samples in absence of any sensitizers under excitation of 980nm laser was observed. The optimal quasi-single crystal rubrene sample with the highest crystallinity shows the strongest UCL at 610nm with up-conversion quantum efficiency of 0.19%. The UCL intensity depends on the proportion of orthorhombic crystal phase, particle size, structural defects, and reabsorption of fluorescence in all samples, while amorphous rubrene sample, such as, both polymethylmethacrylate film dispersed with 0.1wt.% rubrene and 0.5×10−3M rubrene chloroform solution, do not exhibit UCL. According to the dependence of the integral UCL intensity on the excitation power density of 980nm laser, a two-photon absorption feature in our UCL system was obtained. The mechanism of UCL process was also discussed in detail.