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Differential RNA expression is becoming increasingly valuable in evaluating tumor heterogeneity for a better understanding of malignant tumors and guiding personalized therapy. However, traditional techniques for analyzing cellular RNA are mainly focused on determining the absolute level of RNA, which may lead to inaccuracies in understanding tumor heterogeneity, primarily due to i) the subtle differences in certain RNA types that have similar total concentrations and ii) the existence of variations in RNA expression across different samples. Herein, a detachable DNA assembly module is proposed that is capable not only of quantifying the expression level of target RNA but also of innovatively evaluating its proportion within its RNA family population through a sequential assembly and disassembly route. Using the let‐7 family as an experimental model, a significant difference is discovered in let‐7a proportion between normal mammary epithelial cells and breast cancer cells, a characteristic that is often missed in bulk analysis of traditional techniques. By combining concentration and proportion information, the detachable DNA assembly module demonstrates markedly higher efficiency in discerning among various types of cells compared to traditional techniques. This innovative assembly module is expected to offer a new perspective to highlight tumor heterogeneity and guide personalized therapy. [ABSTRACT FROM AUTHOR]