The transition period in dairy cows, encompassing the final weeks of pregnancy through the early stages of lactation, is marked by profound metabolic and physiological changes. This study aimed to evaluate the hematological, biochemical, and metabolic profiles of Sahiwal cows during the transition period in comparison to mid-lactation cows. A total of 24 clinically healthy Sahiwal cows were divided into two groups: Group 1 (transition cows, n = 12) and Group 2 (mid-lactation controls, n = 12). A total of 72 Blood samples were collected at multiple time points from 21 days before calving to 21 days postpartum for hematological, lipid, protein, enzyme, and mineral analyses from group 1 and 12 samples were collected from group 2. Results of the present study indicated significant (P ≤ 0.05) hematological alterations, including reduced white blood cell counts and lymphocyte percentages on the day of calving, along with increased platelet counts. Most of the red blood cell indices (Hb, HCT, MCV, MCH), MCHC, remained within normal physiological ranges. Serum lipid parameters like total cholesterol, triglycerides, HDL-C, LDL-C, and VLDL—declined significantly at calving, reflecting a negative energy balance, and did not return to pre-calving levels even 21 days postpartum. Liver enzymes (AST, ALT, ALP) peaked on the day of calving, possibly due to hepatic stress and muscle damage. Significant reductions in calcium, phosphorus, and magnesium levels were also recorded on the day of calving. Protein metabolism was altered, with reduced total protein and globulin concentrations and increased albumin-to-globulin ratios at parturition. Additionally, elevated levels of blood urea nitrogen and creatinine indicated heightened protein catabolism around calving. In conclusion, the study highlights that Sahiwal cows experience marked metabolic stress during the transition period. These findings underscore the importance of closely monitoring blood biochemical parameters to mitigate health risks and optimize management strategies during this transition period.