Abstract: In order to understand the biofilm formation ability and drug resistance of calf-derived Escherichia coli（E. coli） in Inner Mongolia Autonomous Region, the experiment used morphological observation, biochemical test and PCR method to isolate and identify E. coli, using modified crystal violet staining method and laser confocal inverted microscope identification method, the biofilm formation of calf-derived E. coli was determined. The sensitivity of isolated strains to 20 kinds of antibacterial drugs was determined by micro-dilution method, and the drug resistance spectrum was calculated. The results showed that 51 strains of E. coli were isolated and identified in this experiment. Among the 51 strains of E. coli isolates, the strains with strong biofilm formation ability, medium formation ability, weak formation ability and no biofilm formation ability accounted for 25.5%（13/51）, 17.6%（9/51）, 25.5%（13/51） and 31.4%（16/51）, respectively. The resistant rate of isolates to trimethoprim, sulfadiazine, ampicillin, cephalothin and cefotaxime was 90.2%,78.4%,84.3%,68.6%,68.6%, and sensitive rate of isolates to minocycline, amikacin, cefoxitin and meropenem was 68.6%,88.2%,86.3%,100%. Among E. coli isolates resistant to more than 10 drugs, biofilm-positive strains accounted for 76%, while biofilm-negative strains accounted for only 24%, among which the resistance spectrum of biofilm-positive isolates was wider than that of biofilm-negative isolates. The results suggested that E. coli biofilm-positive isolates from calves were prevalent in Inner Mongolia Autonomous Region, 51 E. coli isolates were resistant to 20 kinds of antibiotics to varying degrees.