[Objective] To analyze the dominant serotype, class I integrons gene cassette, drug resistance characteristics and phylogenetic of Escherichia coli isolated from four large-scale healthy dairy farms in Shandong Province.[Methods] We collected 194 samples of fresh feces from dairy cows in 4 large-scale dairy farms. The isolation and identification of E. coli was tested by using laboratory method, and the serotype was identified by the E. coli diagnostic serums commonly used. The hemolysis of E. coli was detected by 10% sheep blood plate while the sensitivity of 14 conventional antibiotics was detected by K-B method and the ST type of E. coli was analyzed by MLST technique and the cloning relationship among the strains was analyzed by eBURST v3 software.[Results] The results showed that 171 strains of E. coli were isolated from 194 fresh feces samples, mainly pathogenic E. coli (19.9%) and invasive E. coli (17.0%), and the dominant serotypes were O128:K67 (12/171) and O143:K7 (12/171). In addition, the positive rate of hemolytic E. coli was 9.4% (16/171) and the drug susceptibility test showed that the ratio of multi-drug resistant strains was 22.2%, among which the highest resistance rate to ampicillin was 33.9%, followed by tetracycline, which was 24.0%. The results of PCR detection of drug resistance genes and integrons showed that 59.1% of the strains carried the β-lactam resistance gene bla_TEM, 59.1% of the strains carried the aminoglycoside resistance gene ant(2'), and no tetracycline resistance gene (tetA and tetB) was detected. The positive rate of integron I was 4.1% (7/171), dfrA12-aadA2-sul1 was the dominant gene cassette structure (4/171); MLST divided E. coli into 8 ST types, of which ST155 (10/171) and ST58 (45/171) formed a clonal complex and no new ST type was found.[Conclusion] This study confirmed that the dominant serotypes of E. coli isolated from fresh feces in large-scale healthy dairy farms in the region were O128:K67 and O143:K7. Some E. coli were hemolytic and the resistance rate was only high in ampicillin and tetracycline. Moreover, the dominant gene cassette structure is dfrA12-aadA2-sul1. The MLST classification showed that the isolates from different dairy farms were closely related, indicating that the distribution of E. coli in cattle origin was polymorphic, and there was no correlation between serotype and ST type. There is a multi-drug resistance phenomenon in E. coli from dairy cows which is potential dangers of food public safety. This research has certain theoretical guiding significance for improving the safe production and quality evaluation of dairy products in large-scale dairy farms.