Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications.