Biological sex differentiation and hormonal regulation are related with the expression of CYP19 gene in teleost, and so it can be used to explore the relationship between environmental hormone pollution and gene expression. The Gambusia affinis CYP19a cDNA of full sequence was cloned and analyzed for the first time. This would provide comprehensive experimental data for the study of the CYP19 gene as a biomarker for monitoring environmental hormones. Primers were designed according to the conserved region of CYP19a cDNA, and the conserved region was amplified and sequenced. RACE method was used to amplify the G. affinis CYP19a cDNA of full sequence and its protein sequence homology analysis, and the RT-PCR method was used to detect the transcription level of CYP19a mRNA in the sequence. The full length cDNA sequence of G. affinis CYP19a type was cloned. This sequence contains 2020 bp nucleotides and codes 518 amino acids with an open reading frame (ORF) from 238 bp to 1791 bp. We made an analysis of the signal peptide, transmembrane helices, hydrophilic/hydrophobic, primary structure, secondary structure and tertiary structure. When making the homology comparison with CYP19a gene in gonads of other teleost, the gene fragment similarity of mosquitofish were 93%, 84%, 84%, 71%, 71% and 66% with Fundulus heteroclitus, Oryzias laticeps, Rhabdosargus sarba, Carassius auratus, Cyprinus carpio and Danio rerio respectively. This showed that the cloned gene was G. affinis CYP19a gene. Phylogenetic analysis of the CYP19 gene by using MEGA4.0 indicates that CYP19a gene is highly conserved when clustered with other 19 species ovary-derived P450arom gene. We identified the CYP19a cDNA of full sequence is gonadal aromatase gene, and the proof of G. affinis aromatase is by two genes of CYP19a and CYP19b encoding. G. affinis CYP19a has 3 highly conserved fragments, and has catalytic activity.