This thesis concerns the fabrication and modification of novel oxygen and glucose biosensors as well as the application of these biosensors in oxygen and glucose transport research in cell constructs. In Chapter 1, the principle and development of biosensors has been reviewed. Particular attention is paid to oxygen and glucose mass transport research in cell constructs which are crucial for bio-scaffold design in tissue engineering. Chapter 2 details the materials and methods in oxygen and glucose sensor fabrication, modification and characterization. Chapter 3 presents research into practical challenges in oxygen and glucose sensors. For oxygen sensor, membrane biofouling and sensitivity to stirring effect have been detected and successful progresses have been made to reduce their effects. For glucose sensor, membrane biofouling and oxygen tension reliance affect their performance. Remarkable contributions have been made to improve glucose sensors’ stability and reliability. In particular, micro-biosensors have been introduced in the interests of better sensor adaptability for further biomedical applications. Chapter 4 is the experimental section for biosensor applications, and thus provides a detailed description of the cell culture models used in the thesis. Chapter 5 describes the oxygen partial pressure and glucose concentration measurements using biosensors. 2D and 3D cell culture constructs are investigated and results are discussed in this section. It deserves to be mentioned that the modified oxygen and glucose sensor in this thesis are excellent for in vitro biomedical applications, the simultaneously investigation of PO2 and glucose concentration gradient in 3D cell constructs is also a pioneering work in this research field. Chapter 6 illustrates the overall conclusions resulting from the experiments described in the thesis and points out possible future research directions.