Significance We present a site-specific NP labeling strategy for large RNAs by transcription using the TPT3-NaM UBP system. The applicability of this labeling strategy is validated by XSI measurements on 97-nt and 719-nt RNAs with site-specific Nanogold labels. This strategy is generally applicable to any natural and artificial RNAs that can be transcribed by T7 RNA polymerase (RNAP) or covalent conjugation of RNAs with other metal NPs. The usage of a far upstream forward primer during PCR enables easy purification of RNA from DNA templates, and the nondenaturing conditions for conjugation reactions and subsequent purification steps avoids potential large RNA misfolding. This labeling strategy expands our capability to site-specifically conjugate RNAs with NPs for many applications.