Pseudouridine (ψ) is the most abundant RNA modification and plays vital roles in multiple biological processes. However, its biogenesis and functions are not yet fully elucidated. Here we developed an approach for enriching ψ sites by using an exoribonuclease enzyme to eliminate other nucleosides and a new strategy to remove the reads of read-through, coupled with sequencing (exo-ψ-seq) and experimentally and computationally discovered, for the first time, the formation of ψ sites on mRNA can be guided by small nucleolar RNAs (snoRNAs). Moreover, we developed new computational method, psiScan, to identified new ψ sites and interaction between snoRNAs and targets from exo-ψ-seq data. The transcriptome-wide mapping and computational identification of ψ at a single-nucleotide resolution revealed more than 1160 sites, of which 315 sites are dependent on DKC1 enzyme and ~70 sites are further assigned to ~40 H/ACA box snoRNAs. Notably, we demonstrated that DKC1-denpendent ψ modification of RPLX is guided by SNORNA and regulate protein synthesis of RPLX and cell proliferation. Additionally, we found that some orphan SNORNAs leads DKC1 to install the ψ modifications of 28S rRNA. Our work provides a more comprehensive method to detect ψ sites and a powerful experimental method and new computational tool for the future comprehensive research of ψ functions and biogenesis.