To characterize and analyze the structural presentation of a new denture base copolymer with a spiro-acetal cross-linker at 10 and 20 wt.% concentrations by nuclear magnetic resonance (NMR) and field emission scanning electron microscopy-energy-dispersive X-ray (FESEM-EDX) spectroscopies. Also, to evaluate the glass transition temperature (TG) of the new copolymer. The investigational groups G10 and G20 were heat-cured with the new spiro-acetal cross-linker at the above-mentioned concentrations, respectively. The control group G0 was heat-cured without the new cross-linker. Nuclear magnetic resonance and EDX spectroscopies determined the copolymerization along with elemental composition. The surface characteristics were discerned by FESEM. Differential scanning calorimetry was employed to evaluate the TG of the resultant copolymer. Appropriate statistical operations were performed to compare the mean TG of the groups. The new copolymer's structure with the spiro-acetal cross-linker was configured with protons, carbons, aluminum, zirconium, yttrium, and silicon atoms. The TG of the resultant copolymer was high when compared with the G0. The 20 wt.% spiro-acetal cross-linker in the copolymer exhibited the highest TG. The spiro-acetal cross-linking comonomer incorporated in the heat-cure denture polymer produced a new denture base copolymer with elevated TG. The resultant configuration of the new copolymer was characterized, structurally presented, and confirmed. The new copolymer might exhibit augmented strength due to the copolymerized spiro-acetal cross-linker. Moreover, the smooth and regular surface of the copolymer would have minimum or negligible microbial adhesion due to the hydrophobicity of the spiro-acetal comonomer incorporated in the denture base composition. How to cite this article: Ravivarman C, Ajay R, Saatwika L, et al. Structure, Surface Topography, and Glass Transition Temperature of Dental Poly (Methyl Methacrylate) Resin Conjugated with 3,9-bisethenyl-2,4,8,10-tetraoxaspiro [5,5] Undecane as Cross-linker: An In Vitro Research. J Contemp Dent Pract 2024;25(5):486-493.