Abstract: Objective To investigate the biological characteristics of paclitaxel-resistance ovarian cancer cell line and to explore the long term cryopreservation condition, in order to establish stable models in vitro. Methods The drug-sensitive ovarian cancer cell line SKOV3 was given a high dose of impulse and exposed to stepwise increasing doses of paclitaxel, to establish paclitaxel-resistant cell lines, SKOV3/TAX300 and SKOV3/TAX30. The IC50 of paclitaxel-resistant cells were determined by MTT assay. Colony formation, cell growth curve, and doubling time were compared between drug-resistance cells and drug-sensitive cells. The expression genes were analyzed through real time quantitative RT-PCR. The long term cryopreservation condition of drug-resistant cells was determined. Results Two paclitaxel-resistance cell strains, SKOV3/ TAX300 given a high dose of impulse and SKOV3/TAX30 given a stepwise increasing dose of paclitaxel, were developed successfully; the resistant indices were 4.60 and 51.31, respectively. Drug-resistant cells became larger, irregularly shaped, and had longer doubling time, compared with parental cells. Overexpression of multidrvy resistance gene1(MDR1) was detected in both SKOV3/TAX300 and SKOV3/TAX30 cells, while overexpression of lung resistance-related protein(LRP) and protein kinase C alpha(PRKCA) was observed only in SKOV3/TAX300 cells. Low expression of BCL-2-like1(BCL2L1) was seen in SKOV3/TAX30 cells. Drug-resistant cells maintained drug-resistant character better when cryopreserved in the culture medium with proper concentrations of the drug. Conclusion Two SKOV3 paclitaxel- resistant cell lines, SKOV3/TAX300 and SKOV3/TAX30, were induced successfully. These could be used as ideal models in the research of drug resistance. Biological characteristics differed between the two drug resistant cell lines. Drug-resistant cells preserved with the drug could better maintain drug-resistant character.