<p>This study introduces a novel concept, biological in vivo three-dimensional (3D) dose distribution verification, aimed at investigating how respiratory motion affects the efficacy of lung cancer radiotherapy, representing an evolution from the current standard of rigid-body dose distribution verification. A 3D ex vivo biological lung motion simulation device (3D-BioLungEx) was designed to replicate human respiration. A radiotherapy plan of the patient was copied to the porcine lung, which was driven by 3D-BioLungEx to simulate various respiratory patterns that occur during treatment. To ensure anatomical consistency with the patient’s lung structure, during transmission, skin, skeleton, and organs were adjusted according to CT images of the porcine lung. The patient’s radiotherapy plan was then adapted to the porcine lung using the Monaco treatment planning system (TPS). Next, an iterative optimization and scatter inversion-based dose distribution retro-analysis algorithm (IOSI-BLDose) was developed to calculate the dose distribution during treatment. Gamma passing rates were used to quantify discrepancies between this dose distribution and that of the radiotherapy plan. When respiratory conditions were replicated, the passing rate reached up to 93.61%, while irregular breathing dropped it to 70%–90%, primarily due to amplitude changes. However, cycle variations had minimal impact. Compared to conventional rigid-body dose distribution verification, our method provides real-time biological feedback and more effectively captures motion-induced deviations. Accordingly, our biological in vivo 3D dose distribution verification has potential for improving treatment precision and enabling adaptive radiotherapy in clinical practice.</p>