We evaluated the distribution pattern of tannin in 164 sorghum breeding lines and the inhibition rates of amylase, protease, and lipase in sorghum lines with different tannin concentrations. Tannin was existed in the testa of sorghum grain. The tannin content in whole grain of Nampungchal sorghum was 11.54 mg/g, and that in grain (milling rate 73%) and bran fractions was 4.57 mg/g and 28.71 mg/g, respectively. The inhibition rate of α-amylase, α-glucosidase, and β-glucosidase in sorghum lines with tannin was higher than that in sorghum lines without tannin. The inhibition rate of α-glucosidase was greater than 97% in sorghum lines with tannin. The inhibition rate of protease ranged from 20% to 70% in the sorghum lines, showing no discernable trends in tannin content. Lipase inhibition was either very low or not observed and did not seem to correlate with tannin concentration.