Objective To develop a microfluidic device with the adjustable concentration and pressure gradient for 3D cell culture in hydrogel and set up an in vitro model with the capability to closely simulate in vivo microenvironment for cell growth. Methods The microfluidic chip, with a middle channel for 3D cell culture and two side channels for delivering cell culture medium, was designed and fabricated using standard soft lithography and replica molding techniques. Its capability to generate concentration gradient, interstitial flow and image cell in situ was demonstrated. Results A simple microfluidic chip for 3D cell culture in hydrogel with the capability to generate the concentration and pressure gradient was obtained. At a flow rate of 2 μL?min-1 in each side channel, the concentration gradients remained constant after 3 h. The interstitial flow across the gel scaffold was generated by a 100 Pa pressure difference between two-side channels with the pressure gradient of 0.11 Pa/μm. Human adult dermal microvascular endothelial cells (HMVEC) were maintained in 3D culture with collagen type I and observed with confocal microscopy. Conclusions The microfluidic chip is simple and easy to operate and it can simulate the complicated microenvironment in vivo. The chip also allows the multiparameter control of microenvironment, facilitating the better understanding of interaction between cells and microenvironment.