To investigate the drug resistance and virulence of Helicobacter pylori (H. pylori) strains, and explore their relationship with the infection characteristics and biofilm formation ability, gastric mucosa biopsy samples and related clinical data were collected from Huadong Hospital Affiliated to Fudan University between December 2014 and June 2015. H. pylori strains isolated from these clinical samples were subjected to drug resistance assay, virulence gene assay and biofilm-forming assay. Twenty-eight H. pylori strains were acquired. The resistance rates to levofloxacin (LEV), metronidazole (MTZ), clarithromycin (CLA) and amoxicillin (AMX) were 32%, 75%, 11% and 0, respectively. The single and double resistance rates were 61% (17/28) and 36% (10/28), respectively. The detection rates of virulence genes cagA, oipA and vacAs1 were 100%, and no vacAs2 was detected. vacAs1m2 genotype accounted for 61% (17/28), followed by vacAs1m1(11/28, 39%). The prevalence rates of iceA1, iceA2 and iceA1 iceA2 were 54% (15/28), 21% (6/28), 25% (7/28), respectively. The detection rate of dupA was 36% (10/28). The biofilm-forming ability of 28 strains varied. Single factor and independent sample t test analysis showed that strains isolated from 45-59 years old patients, iceA1^＋ dupA^－ genotype, metronidazole-sensitive strains could form stronger biofilm. In conclusion, the resistance rate of H. pylori to metronidazole was highest in single resistance, and the double resistance should not be neglected. The main genotypes of H. pylori isolates were cagA, oipA, vacAs1m2. The H. pylori biofilm-forming ability is related to the age of patients. Specifically, strains isolated from the age 45-59 group have stronger biofilm-forming ability than those obtained from other age groups. In addition, isolates carrying the virulence gene iceA1 have strong biofilm-forming ability. And there is a negative correlation between dupA genotype or metronidazole resistance and the biofilm-forming ability of the H. pylori isolates.