High‐quality spermatozoa are essential for intracytoplasmic sperm injection. However, there is still a lack of noninvasive, objective tools that provide biochemical characteristics to select high‐quality spermatozoa in clinical settings. In this work, a facile yet efficient approach was proposed, rendering the live sperm cell with its head being attached on the surface of a substrate, which allows the subsequent micro‐Raman‐based biochemical analysis and evaluation of sperm DNA integrity noninvasively and nondestructively. Moreover, our preliminary results show that Raman peaks at 1,050 and 1,095 cm−1, which are assigned to DNA backbone mode and whose spectral intensity ratio are suggested as a potential spectral biomarker for evaluation of sperm DNA integrity, can be clearly observed, thus implying our facile yet efficient technique for further potential DNA integrity assessment of live sperm cell. [ABSTRACT FROM AUTHOR]