Immunoassays have become essential tools for detecting infectious viruses. However, traditional monoclonal antibody-dependent immunoassays are costly, fragile, and unstable, especially in complex media. To overcome these challenges, we have developed cost-effective, robust, and high-affinity nanobodies as alternatives to monoclonal antibodies for rapid detection applications. We engineered dual-epitope nanobody (NB) pairs and incorporated them into a sandwich immunosensor design to detect transmitted rotaviruses in rectal swabs and wastewater samples. To further enhance sensitivity, we synthesized an advanced two-dimensional material, MXenes@CNTs@AuNPs, which offers an extensive specific surface area that supports the enrichment and immobilization of NBs. This integration with catalase-modified magnetic probes facilitates signal generation. Subsequently, our sensor achieved a detection limit of 0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming commercial antigen kits with a sensitivity enhancement of 3.77 × 105-fold. The exceptional sensor performance extended to specificity, repeatability, stability, and accuracy across various sample types, establishing it as a promising tool for rotavirus detection. This research outlines a viable strategy for creating a robust and ultrasensitive analytical nanoprobe, thereby addressing the critical need for efficient and reliable viral detection methods in various environments.