The dynamic landscape of mRNA technology highlights the need for innovative quality control (QC) strategies. In this study, we described an efficient one-step digestion approach for concurrent generation of 5'- and 3'-end fragments, enabling simultaneous mRNA capping and poly(A) tail analysis. Tailored 10-23-type DNAzymes, designed from 5'- and 3'-Untranslated Regions (UTRs), selectively cleaved mRNA to release both the 5'-Capped or uncapped short fragments and 3'-Poly(A) tail cleavage products. Polyacrylamide gel electrophoresis (PAGE) and ion pair reversed-phase liquid chromatography (IP-RP LC) analyses confirmed the production of 5'- and 3'-cleavage fragments in a single-step reaction, and LC-mass spectrometry (LC MS) validated these findings. The DNAzyme-mediated cleavage offers notable advantages over other assays for mRNA cap and tail characterization. Direct and simultaneous analysis of both capping efficiency and poly(A) tail length post-cleavage by DNAzymes, without additional purification steps and costly MS analysis, markedly streamlines the sample preparation and analysis process, making it highly suitable for QC testing.