Abstract: The reaction conditions for Double-antibody Sandwich (DAS) ELISA are optimized to detect insecticidal protein in transgenic cry8Ca tobacco against Rutelidae larvae (Coleoptera:Scarabaeidae). As follows: the density of anti-rabbit immun serum and the enzyme(HRP)-labelled antibody were 1:3200 and 1:400 respectively, extracting buffer was pH 9.6 NaHCO3- Na2CO3, coating buffer was pH7.4 PBS, confining liquid was 0.5% Gelatin-PBST, TMB substrate action time was 15min. Cry8Ca protein in transgenic tobaccoes were detected by this DAS –ELISA. The roots of line S12 contain 12.683-14.461ug Cry8Ca protein per gram fresh weigh about 0.052%-0.062% of total protein more than others.