Objective To systematically examine the hematological physiological parameters, serum biochemical indices, and electrocardiographic parameters in mice with a site-specific knock-in of the hSCARB2 gene at the Rosa26 locus (hSCARB2 KI) constructed using CRISPR/Cas9 technology, and to analyze the effects of sex and genotype on these indicators, thereby providing foundational data for the application of this model in enterovirus-related research. Methods Ten 8-week-old hSCARB2 KI mice and ten wild-type mice, each with an equal number of males and females, were selected. Venous blood was collected to measure 18 hematological physiological parameters and 15 serum biochemical indices, and electrocardiograms were simultaneously recorded. An independent samples t-test was used to compare differences between sexes and between genotypes. Results No sex differences were observed in the 18 hematological parameters measured in hSCARB2 KI mice (P>0.05). Among the 12 biochemical parameters, only albumin (ALB) was slightly higher in male mice than in females (P < 0.05). Compared with wild-type mice, hSCARB2 KI mice showed decreased white blood cell count (WBC) and lymphocyte count (Lymph) (P < 0.05), and increased hemoglobin (HGB) concentration (P < 0.01). Among the biochemical indices, aspartate aminotransferase (AST) was decreased (P < 0.01), creatinine (CREA) was increased (P < 0.01), and total bilirubin (TBIL), total bile acids (TBA), and lactate dehydrogenase isoenzyme 1 (LDH1) were increased (P < 0.05). There were no significant differences in electrocardiogram parameters or heart rate between the two groups (P > 0.05). Conclusion Site-specific knock-in of the hSCARB2 gene did not cause significant organic damage in mice. It only induced minor changes in hematological parameters and physiological adaptive adjustments in biochemical indices related to lipid metabolism and renal excretion, with no significant impact on basal cardiac electrophysiological function. This model exhibits stable physiological characteristics and can provide a reliable experimental animal platform and foundational data to support research on enterovirus infection mechanisms, vaccine evaluation, and antiviral drug screening.