The effect of preparation and storage parameters on the number, size and stability of microbubbles covered with bovine serum albumin (BSA) was investigated. A large number of microbubbles with a high stability was obtained at protein concentration of 7.5% or higher, at pH between 5 and 6, at an ionic strength of 1.0 M and at a preheating temperature of 55–60 °C. Microbubbles stored at 4 °C were more stable than those stored at room temperature. This was observed for a specific commercial BSA batch. We found that optimal preparation parameters strongly depend on the batch. Certain BSA batches were found not to lead to microbubbles at all. Microbubbles made with different protein concentration and preheating temperatures shrunk in time to a radius between 300 nm and 350 nm, after which the size remained constant during further storage. We argue that the constant final size can be explained by a thickening of the microbubble shell as a result of the microbubble shrinkage, thereby withstanding the Laplace pressure. The effects of protein concentration, pH and ionic strength on the number of microbubbles directly after sonication can be ascribed to the influence of these parameters on the adsorption speed and ability to cover the surface of air bubbles formed during sonication with enough protein to stabilize the bubble against coalescence and dissolution. We suggest that the effect of temperature during sonication on the formation of microbubbles can be related to thermally induced protein–protein interaction at the air–water interface.