In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies. Therefore, this study produced a recombinant hyaluronidase from Tityus serrulatus scorpion venom, designated as rTsHyal-1, in the Pichia pastoris system. Thus, a gene for TsHyal-1 (gb|KF623285.1) was synthesized and cloned into the pPICZ alpha A vector (GenScript Corporation) for heterologous expression in P. pastoris. rTsHyal-1 was expressed in laboratorial scale in a buffered minimal medium containing methanol (BMM) for 96 h with daily addition of methanol. Expression of rTsHyal-1 resulted in a total protein yield of 0.266 mg/mL. rTsHyal-1 partially purified through cation exchange chromatography presented a specific activity of 1097 TRU/mg, against 838 TRU/mg for the final expressed material, representing a 1.31-fold purification. rTsHyal-1 has molecular mass of 49.5 kDa, and treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, de novo sequencing of rTsHyal-1, performed in MALDI-TOF and Q Exactive Orbitrap MS, resulted in 46.8% of protein sequence coverage. rTsHyal-1 presents the highest substrate specificity to hyaluronan followed by chondroitin-6-sulfate, chondroitin-4-sulfate, and dermatan sulfate and showed an optimum activity at pH 6.0 and 40 A degrees C. These results validate the biotechnological process for the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpion venoms expressed in the P. pastoris system with preserved enzyme activity.