Abstract: PCR and Genome Walking methods were applied to obtain a 168 bp α-amylase gene exon Ⅰ sequence and a 2 063 bp α-amylase gene 5′ flanking sequence from grass carp (Ctenopharyngodon idellus). The sequence alignment of α-amylase gene exon Ⅰ between grass carp and other fishes showed that the similarity was 68%~86%. The sequence analysis of α-amylase gene 5′ flanking sequence of grass carp confirmed a transcription initiation region and a transcriptional start site (TSS). There were a TATA-box at 30 bp and a CAAT-box at -58 bp. There were some transcriptional factor binding sites in the α-amylase gene 5′ flanking sequence, such as GATA-1, OCT-1, GR, HNF-3, AP1 and SP1. The α-amylase gene 5′ flanking sequence of grass carp was cloned successfully, which provides references for studies on differences of α-amylase gene 5′ flanking sequence between different feeding fishes, as well as on the expression, function and regulation mechanism of α-amylase gene in fishes.