||A protease-producing bacterium was isolated and identified as Bacillus sp. TKU004. It thus can be used for deproteinization of squid pen in the preparation of β-chitin. The optimized condition for protease production was found when the culture was shaken at 30 °C for 4 days in 100 mL of medium containing 2% squid pen powder (SPP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4. Under such condition, both the production of protease by Bacillus sp. TKU004 and the resulted protein removal of squid pen attained the optimum. They were 0.065 U/mL and 73%, respectively. An extracellular protease was purified from the culture supernatant of Bacillus sp. TKU004. The molecular weight of TKU004 protease was 27 and 57 kDa assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The optimum pH, optimum temperature, pH stability, thermal stability, Km, Vmax, and activation energy of TKU004 protease were 6–8, 60 °C, pH 5–8, 50 °C, 2.98 mg/mL, 0.14 U/mL, 8.31 J/(mol K), respectively. It was concluded that TKU004 protease is a Zn-containing metalloenzyme, and its dimeric structure contains at least one disulfide bond. The unique characteristic of TKU004 protease is that it retained more than 60% of its original activity after preincubation for 10 days at 25 °C in the presence of tested organic solvents (25%, v/v). This is also the first report of a strain being able to use squid pen as the sole carbon/nitrogen source for proteases production and being used for the reclamation of β-chitin from squid pen.