Biopharmaceutical protein production plays an increasingly important role in human health, including the treatment and prevention of cancer and immune diseases. With the rapid development of synthetic biology technology, cell-free protein synthesis (CFPS) has been regarded as a powerful tool to manufacture a variety of proteins because of its remarkable advantages over living-cell protein synthesis. Working with an efficient and easily scalable reactor, CFPS can meet the requirements of flexible on-demand protein synthesis. To achieve this goal, a new continuous-flow CFPS platform was developed using a tube-in-tube reactor in this study. Compared with other reported reactors, this tube-in-tube reactor has several advantages, such as allowing rapid oxygen diffusion into the reaction substrate to precisely control the oxygen concentration and easy integration with protein purification for continuous-flow protein synthesis. By providing a high dissolved oxygen concentration, the protein concentration reached 3 mg mL⁻¹ with only half of the reaction time in the batch reaction mode. Furthermore, the effects of reaction temperature and residence time of the reaction substrate on protein production were explored. Four representative biopharmaceutical proteins, including antibodies, vaccines, virus-like particles and antimicrobial peptides, were produced to verify the applicability and high efficiency of this tube-in-tube reactor in diverse protein synthesis systems.