In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA). The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins. After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested. After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA. In our conditions, more than 450 mg of EGFP were obtained in 15 h. On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP.