Objectives: To explore the relationship between aldehye dehydrogenase-1 (ALDH-1) and biological characteristics of the stable ALDH-1 knock-down Hela cell lines. Materials and Methods: Transfected Hela cells with lentiviral vector were utilized and puromycin was used to screen and achieve the stable cell lines. The interference efficiency was calculated by performing qRT-PCR to detect the expression of ALDH-1 of three groups including the interfering group, the negative control (NC) group, and the normal Hela group. CCK-8 assays were used to detect the OD value of each group. The coloning formation rate of each group was detected by colony formation assay. cell cycle distribution and apoptosis of each group were also detected by employing cell cycle and apoptosis experiments. Results: The stable ALDH-1 knock-down Hela cell lines were successfully obtained after two weeks' screening; compared with the NC and Hela group, the ALDH-1 expression level of interfering group was 1.05 ± 0.10 (both p < 0.05), whose silencing efficiency was 80.59%. CCK-8 assays verified that the mean OD value of interfering group was lower than that of NC and Hela group. Additionally, colony formation assays showed that the coloning efficiency of interfering group was lower than that of NC and Hela group. Cell cycle experiments proved that the proportion of G0/G1 phase of interfering group was higher than that of the other two groups, while the proportion of S phase was lower. Cell apoptosis assays indicated that the apoptosis rate of interfering group was the highest. Conclusions: Constructing stable interfering Hela cell lines with lentiviral vectors was successful and worthy of promotion. ALDH-1 plays an important role in promoting cell growth and proliferation, maintaining cell cycle and inhibiting Hela cell apoptosis.