Objective: Studying the optimal detection conditions of surface-enhanced Raman Spectroscopy (SERS) for single-stranded deoxyribonucleic Acid (ssDNA).Method: ssDNA GO18(AACCTTTGGTCGGGCAAGGTAGGTT(5′~3′))is used as an example to explore the influence of different detection conditions (concentration of silver sol, laser power and integration time, sample loading method, dosage of coagulant MgSO4) on the results of SERS detection. Under the optimal detection conditions, the stability, repeatability, and detection limit were investigated, and the peak positions of the spectra were assigned.Result: The best conditions for determining ssDNA were as follows: silver sol was concentrated 100 times, laser power was 30% (60 mW), integration time was 30 s, the sample was loaded in 1.0-0.8 high-purity quartz capillary and the amount of MgSO4 (10mM) was 2.0μL. The measurement results under these conditions have high stability and good repeatability, and the detection limit is 0.625μM.Conclusion: This study optimized SERS detection conditions for ssDNA and provided a method basis for further expanding and promoting the application of SERS technology in the field.