Two-photon polymerization (TPP) microfabrication is an advanced technology to fabricate precise three-dimensional (3D) hydrogel micro/nanostructure. 3D hydrogel microstructures fabricated by TPP with sophisticated details and appropriate stiffness are able to effectively simulate the microenvironment used in tissue engineering and drug delivery. The mechanical property of the microstructures, for instance, the Young's modulus is crucial to achieve the microstructures with high fidelity. In this study, the mechanical property of the poly(ethylene glycol) (PEG) 3D microstructures fabricated with various laser powers, writing speeds and layer distances in the air was investigated by characterizing the Young's modulus. Meanwhile, the Young's modulus of the microstructure with different layer distances in water was determined as 3.50-6.52 MPa. Furthermore, 3D PEG microstructures simulating red blood cell morphology of different postures and sizes were successfully fabricated. (C) 2017 Elsevier B.V. All rights reserved.