The membrane based on receptor functionalization provides a new paradigm for the development of mechanical biosensors. However, improvement of sensitivity and test accuracy is still a challenge for mechanical biosensors in practical application. Herein, a surface stress mechanical biosensor (MBioS) based on double‐side‐gold nanoparticale (AuNP)‐coated grid‐type polydimethylsiloxane (PDMS) membrane (D‐G‐MBioS) and 3D printing for human serum albumin (HSA) detection is developed. The surface stress is amplified by the grid coupling sandwich immune structure to improve the sensitivity of the MbioS, successfully reducing limite of detection (LOD) by two orders of magnitude. By self‐assembly of AuNPs, the double‐side‐coated PDMS membrane is prepared, and the detection accuracy of the sensor is improved. The 3D printing is employed to improve the consistency of the MBioS. The D‐G‐MBioS system is constructed, realizing the HSA detection in range of 0–50 µg mL−1 with a LOD of 20.6 ng mL−1. In addition, the D‐G‐MBioS based on sandwich assay is exhibited the high specificity, selectivity, stability, and repeatability. Especially, the D‐G‐MBioS can detect the HSA in real samples. This study provides a way to improve the sensitivity of mechanical biosensor.