We propose a single-shot lens-free phase retrieval (SSLFPR) method in a lens-free on-chip microscopy (LFOCM) system based on a partially coherent light emitting diode (LED) illumination. The finite bandwidth (∼23.95 nm) of LED illumination is divided into a series of quasi-monochromatic components according to the LED spectrum measured by a spectrometer. When the "virtual wavelength scanning" phase retrieval method is combined with the dynamic phase support constraint, the resolution loss caused by the spatiotemporal partial coherence of the light source can be effectively compensated. At the same time, the nonlinearity characteristics of the support constraint help to further improve the imaging resolution, accelerate the convergence of the iteration process, and greatly eliminate the artifacts. Based on the proposed SSLFPR method, we demonstrate that the phase information of samples (including phase resolution target and polystyrene microspheres) illuminated by a LED can be accurately retrieved based on one single diffraction pattern. The SSLFPR method has a half-width resolution of 977 nm across a large field-of-view (FOV) of 19.53 mm2, which is 1.41 × the resolution of the conventional approach. We also imaged living Henrietta Lacks (HeLa) cells cultured in vitro, further demonstrating the real-time single-shot quantitative phase imaging (QPI) capability of SSLFPR for dynamic samples. Given its simple hardware, high throughput, and single-frame high-resolution QPI capability, SSLFPR is expected to be adopted in a wide range of biological and medical applications.