Presented herein is a novel headspace single drop microextraction (HS-SDME) based on temperature gradient (TG) for an on-site preconcentration technique of volatile and semivolatile samples. First, an inner vial cap was designed as a cooling device for acceptor droplet in HS-SDME unit to achieve fast and efficient microextraction. Second, for the first time, an in-vial TG was generated between the donor phase in a sample vial at 80 °C and the acceptor droplet under the inner vial cap containing cooling liquid at -20 °C for a TG-HS-SDME. Third, a simple mathematic model and numerical simulations were developed by using heat transfer in fluids, Navier-Stokes and mass balance equations for conditional optimization, and dynamic illumination of the proposed extraction based on COMSOL Multiphysics. Five chlorophenols (CPs) were selected as model analytes to authenticate the proposed method. The comparisons revealed that the simulative results were in good agreement with the quantitative experiments, verifying the design of TG-HS-SDME via the numerical simulation. Under the optimum conditions, the extraction enrichments were improved from 302- to 388-fold within 2 min only, providing 3.5 to 4 times higher enrichment factors as compared to a typical HS-SDME. The simulation indicated that these improvements in the extraction kinetics could be attributed due to the applied temperature gap between the sample matrix and acceptor droplet within the small volume of headspace. Additionally, the experiments demonstrated a good linearity (0.03-100 μg/L, R2 > 0.9986), low limit of detection (7-10 ng/L), and fair repeatability (<5.9% RSD, n = 6). All of the simulative and experimental results indicated the robustness, precision, and usefulness of TG-HS-SDME for trace analyses of analytes in a wide variety of environmental, pharmaceutical, food safety, and forensic samples.