Effect of lipid removal, extraction medium and extraction time on the isolation and recovery of trypsin inhibitor from yellowfin tuna (Thunnus albacores) roe was investigated. Trypsin inhibitor extracted from defatted tuna roe showed the higher specific inhibitory activity than extracted from origin tuna roe. Optimal extraction medium was attained by shaking the defatted yellowfin tuna roe powder in 10 mm Na phosphate buffer (pH 7.0) containing 0.5 m NaCl (P < 0.05). The extraction time affected the inhibitor recovery significantly (P < 0.05). The extraction time of 30 min was optimum for recovery of trypsin inhibitor from yellowfin tuna roe. The biochemical properties of trypsin inhibitor from yellowfin tuna roes were also determined. The inhibitor was stable to heat treatment up to 60C and over a broad pH range (5-8). Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the trypsin inhibitory activity. However, the activity decreased when trypsin inhibitor was incubated with metal ions at ambient temperature for 30 min.