We adopted simultaneously three effects of milling, carbon (C) doping and solid-state reaction to improve critical current properties of MgB2 superconducting wire. The influences of heat-treatment temperature, including a conventional solid–liquid reaction of 650–1000 °C and a solid-state reaction of 600 °C below the melting point (650 °C) of Mg powder, were examined on the transport superconducting properties of in situ powder-in-tube (PIT) processed MgB2/Fe wires using ball-milled and glycerin-treated boron (B) powder. The aims of the mechanical milling and liquid glycerin treatment of the B powder were to reduce the grain size of the MgB2 and achieve homogeneous C incorporation into the MgB2, respectively. The superconducting properties of MgB2 wires heat-treated in the range of 650–1000 °C were investigated, and it was also investigated as to whether the C incorporation occurred even in a low-temperature solid-state process of 600 °C, thus resulting in an improvement of the superconducting properties by obtaining both high grain boundary density and C substitution effects. The MgB2 phase formation, actual C substitution amount, full width at half maximum (FWHM), critical temperature (Tc), magnetic field dependence of transport critical current density (Jc) and temperature dependence of the upper critical field (Hc2) were evaluated for glycerin-doped MgB2/Fe wires fabricated at different heat-treatment temperatures. The glycerin-doped MgB2 wire using milled B powder heat-treated at a solid-state of 600 °C showed the highest transport Jc values at 4.2 K over the entire applied field regime. It was revealed that the grain boundary density was higher and that the C substitution also occurred by a low temperature heat-treatment process, which led to a higher Jc. In addition, a solid–solid diffusion reaction with the pre-treated B powder resulted in poor crystallinity, which enhanced Hc2 and improved Jc.