Background: The development of rapid, sensitive, and environmentally friendly sample preparation methods is important for monitoring antibiotic residues in complex biological matrices. Objective: The objective of this study was to develop a novel microwave-assisted density-tunable dispersive solid-phase extraction (MA-DT-DSPE) method for the extraction and preconcentration of tetracycline from human urine samples prior to high-performance liquid chromatography with diode array detection (HPLC-DAD). Materials and Methods: The proposed method employed a low-density organic sorbent (safranal) dissolved in a volatile dispersive solvent and rapidly injected into the aqueous sample. Microwave irradiation caused instantaneous volatilization of the dispersive solvent, resulting in in-situ formation and homogeneous dispersion of fine sorbent droplets without mechanical agitation. Because the sorbent was less dense than water, the analyte-enriched phase floated to the surface for easy collection. The effects of dispersive solvent type and volume, sorbent mass, microwave power, irradiation time, and ionic strength were systematically optimized. Results: Under optimal conditions, the method showed a low limit of detection of 0.26 ng mL⁻¹, linearity over 0.8–200 ng mL⁻¹ (R² ≥ 0.994), extraction recovery of 74%, and precision better than 5.6% relative standard deviation. Analysis of spiked urine samples gave relative recoveries of 96%–107%, indicating negligible matrix effects. Conclusions: It can be concluded that the MA-DT-DSPE-HPLC-DAD method is a rapid, simple, sensitive, reproducible, and eco-friendly analytical approach, making it a promising alternative for routine determination of tetracycline residues in human urine samples.