The expansion of the canonical genetic alphabet via the Artificially Expanded Genetic Information System (AEGIS) offers opportunities in molecular diagnostics, therapeutics, and synthetic biology. However, current sequencing and detection methods are often bottlenecked by biophysical challenges, such as signal degeneracy and the lack of high-resolution, label-free sensing for synthetic nucleobases. Density functional theory calculations with van der Waals corrections and electronic transport simulations reveal that Janus MoSSe monolayers act as a versatile platform for the label-free detection of expanded genetic alphabets. They differentiate natural and synthetic AEGIS nucleobases through distinct adsorption and electronic signatures, providing a potential pathway for sequencing DNA, RNA, and other artificial polymers. Nucleobases primarily adsorb through physisorption with stronger binding on the selenium-terminated surface than the sulfur-terminated side. Adsorption modulates the conductivity and transmission properties of MoSSe in a base-specific way, enabling electronic differentiation. Synthetic xenonucleobases such as J and natural bases such as G show the strongest binding and electronic coupling, while bases like T display minimal interaction. Charge transfer and band-structure analyses indicate pronounced p-type doping effects on the sulfur side via midgap states with more subtle perturbations on the selenium side. Nonequilibrium Green’s function (NEGF) transport calculations highlight base-dependent transmission and current differences, suggesting MoSSe’s potential for highly selective and sensitive molecular identification. These findings establish Janus MoSSe as a promising candidate for next-generation biosensors and a complementary approach for reading expanded genetic alphabets.