The longitudinal beam dimensions in permanent structures within large cross-sectional underground spaces constructed by the combined pipe-jacking and three-circle shield method are too large, leading to compressed spaces under the beam. To address this problem, a novel type of combined structure—prestressed segment-steel reinforced concrete composite beam—is proposed. Six working conditions are set with and without prestressed reinforcement and prestress to conduct flexural tests on the failure mode, stress performance, deformation capacity, and strain patterns. The primary conclusions are as follows: (1) Under positive and negative bending moments, prestressed composite beams exhibit ductile damage. (2) Under positive and negative bending moments, prestressed reinforcement significantly improves the ultimate bearing capacity of such combined beams, increasing it by 69.3% and 12.1%, respectively. (3) Under positive bending moments, prestressed reinforcement slightly reduces the cracking load but significantly improves the deformation resistance of such combined beams. Under negative bending moments, prestressed reinforcement significantly improves the crack and deformation resistance of such combined beams. (4) Increasing prestress slightly improves the ultimate bearing capacity, crack resistance, and deformation capacity of the combined beams under positive bending moments while substantially enhancing these properties under negative bending moments. (5) The strain in the mid-span section is linearly distributed along the height direction of the beam, consistent with the assumption of flat section.