Polycation-based mRNA delivery systems have an issue with mRNA integrity in the physiological milieu, particularly in blood compartments, despite their vast potential in mRNA therapeutics. Without comprehensive mechanistic analyses, design concepts of polyplexes for in vivo use remain unclear. Herein, we systematically assessed several potential design parameters of polyplex stabilization and provided mechanistic insight into the processes of mRNA degradation loaded in polyplexes, focusing on RNase attack, a process believed to be the leading cause of loss of mRNA integrity loaded into polyplexes. For this purpose, polyplex micelles (PMs) from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymer were used as a platform polyplex system feasible for in vivo application. Elongating PEG from 12-kDa to 42-kDa failed to improve RNase stability despite a plausible increase in the PEG layer thickness on the PM surface. Meanwhile, the elongation of polycation segments and a subtle but critical modulation in the side chain of polycation structure, i.e., changing from poly(l-lysine) to poly(l-ornithine), significantly improved the resistance of cargo mRNA against RNase attack. Nonetheless, nearly 50 % of mRNA was degraded even in the optimal PM formulation after 30 min incubation in 50 % serum. Plausible mechanisms of mRNA degradation include (i) dissociation of PM structure by polyion exchange reaction with anionic biomolecules in serum to release mRNA, followed by RNase attack and (ii) RNase penetration into PM interior to directly attack cargo mRNA without PM dissociation. A series of mechanistic experiments revealed that mRNA was still settled in the PMs even after a loss of mRNA integrity by 50 % serum treatment, indicating the latter to be the main reason for the degradation of cargo mRNA. Further, the integrity of PM structure and cargo mRNA in circulating blood was evaluated separately in mice. Intravital microscopic observation of mRNA complexation status using fluorescence resonance energy transfer (FRET) indicates prolonged mRNA retention in the PM structure even under blood circulation. In contrast, quantitative PCR-based evaluation of mRNA integrity revealed the occurrence of prompt mRNA degradation in the same condition. This study highlights that PM structure is robust enough against dissociation under blood circulation. Yet, the remaining challenge toward optimizing PM-based mRNA delivery systems for systemic application is to build a functionality to prevent RNase invasion into the polyplex core storing cargo mRNA.