Acrolein holds excellent potential as a critical biomarker in various oxidative stress-related diseases, and direct measurement of acrolein in biological systems is becoming essential to provide information for diagnosis and therapeutic purposes. In this review, we will discuss some available techniques for the detection of acrolein from biological samples. A conventional analytical method for the detection of acrolein by using high-performance liquid chromatography analysis after derivatization with 3-aminophenol is available. However, it is not suitable for high-throughput assay and inconvenient for measurement in clinical practice. On the other hand, we have recently discovered that phenyl azide can rapidly and selectively react with acrolein in a click manner to provide 4-formyl-1,2,3-triazoline through 1,3-dipolar cycloaddition. Moreover, we have successfully utilized this acrolein-azide click reaction as a simple and robust method for detecting and visualizing acrolein generated by live cells. Herein, we will describe our reaction-based acrolein sensor and its application to detect and visualize breast cancer tissues. We utilized the azide-acrolein click reaction-based method to discriminate breast cancer lesion from the normal breast gland, which resected from breast cancer patients. This method is the first example of an organic synthetic chemistry-based approach that can be used not only to visualize the cancer tissue but also to distinguish morphology of the resected tissue only within a few minutes. It has a potential clinical application for breast-conserving surgery. Furthermore, the ability to perform chemical reactions with cancer metabolites only at the desired cancer site is highly advantageous for cancer therapy.