A cell culture device includes a main body and a plug element. The main body includes a slot, an open groove and a fluid chamber. The open groove is connected with one side of the slot. The fluid chamber is disposed inside the main body and connected with another side of the slot. The plug element includes a first cell culture chamber and a first porous membrane. The first porous membrane is disposed at one side of the first cell culture chamber. The plug element is detachably plugged into the slot. When the plug element is plugged into the slot, the first cell culture chamber is communicated with the open groove to form an open space, and the open space and the fluid chamber are separated by the first porous membrane.