Recently developed technique of vortex-assisted liquid–liquid microextraction (VALLME) has thus far proven to be efficient for the rapid extraction of trace amounts of organic pollutants with both high enrichment factors and low detection limits. In this paper, a new and easy one-step in-syringe device for VALLME has been proposed and applied for the analysis of three fungicides in real aqueous samples. This novel technique performed extraction, separation, preconcentration, and injection in one step using a 5 mL syringe, thus expanding the level of automation of VALLME. The practicality of the technique was evaluated through the analysis of three fungicides (azoxystrobin, diethofencarb, and pyrimethanil) in aqueous samples by high performance liquid chromatography-diode array detection. 1-octanol was used as the extractant. Under the optimized conditions, the limits of detection and enrichment factors were 0.96 μg L−1, 144 for azoxystrobin, 1.33 μg L−1, 183 for diethofencarb, and 0.73 μg L−1, 176 for pyrimethanil. Good reproducibility expressed as the intra-day and inter-day precisions (RSD) at 40 μg L−1 concentration level were below 4.0% and 6.1% (n = 6). The recoveries of the spiked real water samples at three different concentration levels of 4, 10, 40 μg L−1 ranged from 91.6–116.8%, 81.3–107.3% and 81.4–104.4%, respectively.