Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained significant attention for mRNA analysis. Despite their potential, these methods still face challenges, including false positives and low amplification efficiency. To overcome these limitations, we have developed a confined catalytic hairpin assembly and hybridization chain reaction (CHA-HCR) system that utilizes cholesterol-modified hairpin probes to enhance the sensitivity and specificity of mRNA detection. This system integrates cholesterol-modified hairpin probes (CHA probes) with hybridization chain reaction probes (HCR probes), leveraging hydrophobicity-mediated assembly to create a robust biosensing platform. The CHA-HCR system initiates a complex formation with the target mRNA, triggering a cascade of hybridization events that amplify the fluorescence signal. Employing ANXA2 mRNA as a model system, our results reveal that the CHA-HCR system achieves a detection limit (LOD) of 8.7 pM and offers high selectivity, effectively distinguishing ANXA2 mRNA from similar RNAs with single-base mismatches. Additionally, the CHA-HCR probes demonstrate stable detection performance in complex environments and exhibit excellent sensing capabilities in clinical tissue samples, successfully differentiating ANXA2 mRNA expression between leiomyoma and adenomyosis patient tissues. This study introduces a promising approach for the early diagnosis and monitoring of diseases associated with mRNA, potentially contributing to improved clinical outcomes and personalized treatment strategies.