The invention relates to a construction of a Comamonas testosteroni 3,17beta-HSD gene reinforced strain, and belongs to the microbial field. The construction of the Comamonas testosteroni 3,17beta-HSD gene reinforced strain is characterized in that a recombinant plasmid is pK-Tacpromoter-3,17beta-HSD; and the construction comprises the following steps: carrying out PCR technological amplification, and target segment recovery and purification of a Comamonas testosteroni genome which is a template to connect with a pUCm-T carrier; 2, carrying out PCR technological amplification, and target segment recovery and purification of a plasmid pAH10 which is a template to connect with the pUCm-T carrier; 3, extracting a recombinant plasmid 3,17beta-HSD-T through adopting a column plasmid DNA small-amount extraction kit; and 4, respectively extracting the recombinant plasmid and pK-3,17beta-HSD through adopting the column plasmid DNA small-amount extraction kit. According to the invention, the Comamonas testosteroni 3,17beta-HSD gene reinforced strain is constructed for the first time and is applied, and the estriol degradation efficiency, the cholesterol degradation efficiency and the stosterone degradation efficiency of the Comamonas testosteroni 3,17beta-HSD gene reinforced strain are 30%, 15% and 150% higher than that of a wild Comamonas testosteroni strain respectively.