Cry proteins are biopesticides to control insect pests from several decades. However, spores have short shelf-life and sensitivity to the environmental factors. Hence, genes encoding Cry proteins have been transferred to crop plants through transgenic approach. Detection of expressed Cry protein both quantitatively and qualitatively is important at developmental stage to identify the promising events and during post release into field. The serological assays so far developed for detection were able to provide information for presence or absence of protein. In present investigation, Cry1Ac protein was used an antigen and Single-chain antibody fragments (scFv) polyclonal were developed using human synthetic scFv Tomlinson library (MRC Laboratories, Cambridge, UK). To get specific scFv binders, the library was panned against the immobilized antigens for 4 rounds. The selected scFv polyclonal against Cry1Ac were tested for its sensitivity, specificity, reliability and its use in qualitative assay. Our results indicate that the anti-Cry1Ac polyclonal antibody had: (i) good antibody specificity; (ii) can detect as low as 10 ng/ml Cry1Ac protein; (iii) is not cross-reactive with other Cry proteins. Dot blot assay developed is a rapid, sensitive, cost effective and simple to use, therefore suitable for the detection of Cry1Ac gene expression from transgenic plants during routine assays.