Lactation in dairy cows implies comprehensive endocrine and metabolic changes including a systemic electrolytic reaction. Previous studies have rarely considered these specific demands due to the influence of lactation periods. Therefore, this study aimed to assess the effects of early, middle, and late lactation phases on the dynamic changes in serum concentrations of progesterone (P4), 17β-oestradiol (E2), cortisol, and some electrolytes (Ca++, Mg++, Na+, K+, Cl−, Pi) and biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), total bilirubin, urea, and iron (Fe++) in 10 Holstein, 10 Brown Swiss, and 10 Modicana multiparous healthy dairy cows (4.2 ± 1.7 years of age) sampled at 60-day intervals throughout lactation. Lactation induced significant changes in the concentrations of P4, which peaked at >120–180 days, decreased at >240–300 days, and increased again after 300 days. Cortisol showed an opposite trend to P4, with concentrations progressively decreasing, except for the phase between >240 and 300 days, and a steep drop at >300 days compared to previous phases. Na+ concentrations showed the lowest values at 0–60 d and the highest ones at >180–240 days, whereas Mg++ showed the highest values at >60–120 d and the lowest at >300 d. Significant correlations were found between P4 with cortisol, Cl− and K+, and cortisol with Ca++ and LDH. Significant differences in average concentrations of AST, ALT, LDH, Ca++, Mg++, and Fe++ were observed among different dairy cow breeds. Understanding the dynamic changes in hormone levels, electrolytes, and biochemical parameters during different lactation phases, while considering breed differences in dairy cows, is crucial for improving herd health management and milk production in commercial dairy farms.