Acrylamide was selected as a monomer to generate polyacrylamide network in 3D printing mortars via in-situ polymerization, and the effect of in-situ polymerization on the mechanical properties of 3D printing mortars was investigated. The influence mechanism was analyzed by Fourier transform infrared spectroscopy, scanning electron microscopy, mercury intrusion porosimetry, and X-ray computed tomography. The results show that the in-situ polymerization of AM can enhance the fluidity of 3D printing mortars, reduce the generation of macroscopic defects, and improve the printing quality. After AM monomers are polymerized, a “rigid-flexible” composite network structure is formed, in which the organic network and the cement hydration products are intertwined. This structure greatly enhances the flexural strength and relative bonding strength of the 3D printed specimen. The 28 d flexural strength and the relative interfacial bonding strength can be increased by 52.4% and 78.1%, respectively.