BACKGROUND: Most enzymes, including protease, play a key role in biotechnology, but their use is quite limited due to poor recovery, limited reusability and instability. Immobilized enzymes offer advantages over free enzymes. This paper reports a simple method for the preparation of immobilized papain, an endolytic cysteine protease (EC: 3.4.22.2), on carboxyl-activated silica nanoparticles. RESULTS: The carboxyl-activated carriers produced reactive carboxyl groups which then react with the free amino groups of enzyme to give peptide bonds (?CO?NH?). The results showed that the thermal and pH stabilities of the immobilized papain were higher than those of free enzyme. And the apparent Km value of the immobilized papain was 1.26 times higher than that of free enzyme. Moreover, the immobilized papain retained more than 45% of the original activity after ten reuses continuously. CONCLUSION: The results indicated that papain was successfully immobilized on the surface of the activated carriers. The immobilized papain had not only higher activity recovery, but also better stability, reusability and environmental adaptability. Copyright (C) 2012 Society of Chemical Industry