The invention discloses a 3D three-dimensional cell culture system which is applicable to cell experiments and bio-engineering production. The system comprises a cell culture dish, sterile soft hoses, a peristaltic pump, an optical fiber sensor, a signal processing system, a temperature controller, a gas controller, a cell culture solution storage tank, a carbon dioxide storage tank, a computer as well as associated software and a light source. According to a working principle, such information as temperature, pH, cell density and the like in the cell culture dish is monitored by virtue of the optical fiber sensor in real time and the information is analyzed and processed by virtue of the computer, so that related components are controlled to precisely regulate corresponding culture conditions, and various indexes in a cell culture medium are kept balanced and stable. The system can automatically complete such complex experimental operations as cell observation, culture solution replacing, temperature measurement, pH value regulation and the like, manual labor can be replaced, so that manual cost is reduced; and meanwhile, the system, which precisely controls culture conditions, can guarantee the stability and uniformity of experimental factors.