Background: The demand of recycling renewable agricultural by-products is increasing. Radiation breeding is a method used to improve saccharification efficiency. Thus, we investigated the effect of gamma ray irradiation on the pretreatment and enzymatic hydrolysis of the stalks of Senna tora, an important medicinal plants. Methods and Results: S. tora seeds were irradiated with gamma ray at doses of 100, 200, 300, and 400 Gy. In the pretreated biomass, glucan and lignin content were higher in the M1 (1st generations of irradiation) S. tora stalks than in the M2 (2nd generations of irradiation) stalks, this can be explained by the higher degradation rate in M1. After oxalic acid pretreatment, the concentration of total phenolic compounds (TPCs) in the hydrolysate increased in the gamma ray treated seeds. The highest relative increase rate in crystallinity in the pretreated biomass was observed in M1-400 Gy and M2-100 Gy. The cellulose conversion rate was higher in M1 than in M2, except for 200 Gy. Conclusions: Gamma ray irradiation at an appropriate dose can be used to improve the efficiency of pretreatment and enzymatic hydrolysis, thereby increasing biomass availability.