Single-molecule detection using surface-enhanced Raman spectroscopy (SERS) has attracted increasing attention in chemical and biomedical analysis. However, it remains a major challenge to probe single biomolecules by means of SERS hot spots owing to the small volume of hot spots and their random distribution on substrates. We here report an in situ hot-spot assembly method as a general strategy for probing single biomolecules. As a proof-of-concept, this proposed strategy was successfully used for the detection of single microRNA-21 (miRNA-21, a potential cancer biomarker) at the single-cell level, showing great capability in differentiating the expression of miRNA-21 in single cancer cells from normal cells. This approach was further extended to single-protein detection. The versatility of the strategy opens an exciting avenue for single-molecule detection of biomarkers of interest and thus holds great promise in a variety of biological and biomedical applications.