Percutaneous transthoracic needle biopsy (PTNB) is an important modality for the qualitative diagnosis of lung cancer, which relies on medical-image guidance. X-ray equipment features wide availability and low radiation dose; nevertheless, X-ray-guided PTNB requires repeated calibration using physical calibrators. Accordingly, this paper proposes an X-ray-guided lung puncture navigation method integrating the direct linear transformation-Gauss-Newton method (DLT-GNM) and marker-free registration. Preoperatively, initial imaging parameters are acquired via fluoroscopic calibration. Intraoperatively, a binocular camera tracks the spatial pose of the C-arm in real time to virtually reconstruct calibration points. DLT-GNM is employed to solve the projection model and realize marker-free real-time navigation. Respiratory phantom experiments show that the overall localization error is (0.38 ± 0.25) mm under matched respiratory phases. It is verified that this method delivers high accuracy and simple operation, and possesses favorable clinical application potential for cases with large lesions or identifiable targets clearly visualized under X-ray fluoroscopy.