The full length cDNA sequence of β-galactosidase gene named McGAL was cloned by 3’RACE technique based on the related EST sequence from the normalized Full-Length cDNA Library of bitter gourd Fruit. Sequence analysis showed that the nucleotide sequence of the gene is2261bp，containing a 2187 bp open reading frame(ORF) that encodes 719 amino acids. The sequence has been deposited in GenBank database with the accession number of AFD54987.1. The gene contains the putative active site-containing consensus sequence pattern G-G-P-[LIVM]-x-Q-x-E-N-E-[FY] belonging to glycosyl hydrolase family 35 without lectin domain at their C-termini. The McGAL exhibits a typical structure with a membrane -spanning domain. In the secondary structure, α-helix, Extended strand ,β-turn and Random coil are 19.89%, 26.98% , 6.54% and 46.59%, respectively. Amino acid sequence alignment shows the McGAL shows higher identity with Cicer arietimum, Medicago trumcatula, Vigna radiate, Glycina max and Lupimus angustifolius, as 73%, 73%, 73%, 72% and 71%. Subcellular localization revealed the McGAL was located in mitochondrial inner membrane. Fluorescent quantitative PCR analysis shows the McGAL gene expressed the highest levels at the mature green stage and decreased thereafter, which means the gene may be related to early ripening stage.