A solid-phase microextraction (SPME) injection gas chromatography was validated with the flame ionization detection method (GC-FID) using a capillary column to detect ethanol. The method was used to determine ethanol in fluids with biomedical, clinical, and forensic importance, including water, phosphate-buffered saline (PBS), and artificial sweat. The strategy produced good peak resolution and showed a linear correlation between the concentration and peak areas for ethanol in all matrices. The inter- and intra-day precisions of the method were below 15.5% and 6.5%, respectively, varying according to the matrix. The method achieved detection limits below 1.3 mg/L, varying according to the matrix. Lower limits were obtained for the aqueous solution (0.22 mg/L), followed by the PBS solution (0.96 mg/L), and finally, the sweat solution (1.29 mg/L). This method is easy to perform and suitable for use in routine clinical biochemistry and forensic laboratories, allowing ethanol detection at lower concentrations using lower temperature and time of extraction, when compared with other studies.