An ultrasound‐assisted low‐density‐solvent‐based solvent‐demulsified dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography with ultraviolet detection method has been established for the purpose of microextraction and recognition of tyrosine, phenylalanine and tryptophan from plasma and urine samples. For the preconcentration of analytes, 10 mL of sample solution was placed in test tube. A blend of 200 μL 1‐dodecanol and 100 μL acetonitrile were quickly injected into the sample solution and then shaken for 4.8 min using ultrasonic irradiation. The addition of demulsifier into the formed emulsion results in rapid separation of the mixture into two phases. The central composite design was used for optimization of microextraction conditions. Under the optimized conditions, the calibration graphs of tyrosine, phenylalanine and tryptophan were linear in the range of 1–200 μmol L−1. Limits of detection and quantification were 0.17–0.32 and 0.64–1.09 μmol L−1, respectively. This procedure was successfully used to determine target analytes in spiked plasma and urine samples; the relative mean recoveries ranged from 84.7 to 96.4%.