A growing number of studies suggest that epidermal growth factor (EGF) plays an important role in early-weaned animals. The objective of this experiment was to compare the biological activity of intracellularly expressed EGF (IE-EGF), extracellularly expressed EGF (EE-EGF), and tagged EGF (T-EGF) from Saccharomyces cerevisiae (S. cerevisiae) both in vivo and in vitro. Strains of S. cerevisiae expressing IE-EGF, EE-EGF, and T-EGF were designated INVSc1-IE(+), INVSc1-EE(+), and INVSc1-TE(-), respectively. The production performance, intestinal development, physio-biochemical indexes, and immunological function of early-weaned rats were measured in vivo to evaluate the biological activity of IE-EGF, EE-EGF, and T-EGF. In addition, the proliferation of rat enterocyte was also measured in vitro. In the in vivo experiment, the recombinant S. cerevisiae was shown to survive throughout the intestinal tract. The production performance (e.g., body weight) and intestinal development (e.g., mean villous height, crypt depth, total protein, DNA, and RNA) of the rats were significantly enhanced in the INVSc1-IE(+) group compared with the INVSc1-EE(+) and INVSc1-TE(-) groups (P < 0.05). However, the levels of lactate dehydrogenase (LDH), immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) showed no difference in the INVSc1-IE(+) group compared to the INVSc1-EE(+) and INVSc1-TE(-) groups (P > 0.05), with the only significant difference being found for creatine kinase (CK) (P < 0.05). In the in vitro experiment, the proliferation of enterocyte was significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF (P < 0.05). Herein, IE-EGF is more suitable for application to early-weaned animals compared with EE-EGF and T-EGF.