This study was conducted on canine puppies (n=600) with diarrhoea during October 2019-20 in district Lahore, Pakistan to know the molecular prevalence of E. coli O157:H7, its associated risk factors and the impact of bacterial diarrhea on various blood parameters and serum electrolytes. PCR-based positive (n=21) and negative (n=21) cases were sampled for their blood, and serum for the haemato-serum electrolyte analysis.  The results showed that the overall prevalence of E. coli O157:H7 was 3.5% (P<0.05) in canine diarrheic pups. The highest prevalence of E. coli O157:H7 was found within the UVAS pet center (10 %, 10/100). Season, food, drinking water plus animal breed, sex, co-habitation with other animals and contact with animalsˋfeces, were significantly (P<0.05) associated with the E. coli O157:H7  prevalence. Mean value of Hb (P<0.01), RBCs (P<0.05), lymphocytes (P<0.01) and platelets (P<0.05) were reduced significantly, while neutrophils, PCV and TLC were increased significantly(P<0.01) in the infected group as compared to the control healthy group. A significant decrease was found in sodium mEq/l (P<0.01) and potassium mEq/l (P<0.05), while a significant (P<0.01) increase was found in chloride mEq/l and bicarbonate mmol/l in the infected group as compared to control healthy group. In conclusion, the E. coli O157:H7 was present in the canine puppies of district Lahore Pakistan and had a high effect on blood parameters and serum electrolytes.