Cholesterol determination in body is important in diagnosis of diseases like coronary heart disease, arteriosclerosis, diabetes, and obstructive jaundice. This research aims at developing fluorimetric cholesterol biosensors based on self-assembled mesoporous alginate-silica (Algilica) microspheres. For preparing the biosensor, Pt-(II)-octaethylporphine (PtOEP; oxygen sensitive metalloporphyrin) dye has been loaded in the Algilica microspheres using the solvent-mediated precipitation method. Cholesterol oxidase (ChOx) was then covalently conjugated to PtOEP/Algilica microspheres using EDC and NHS reagents. PtOEP dye and enzyme encapsulation, activity and stability were then analyzed. Layer-by-layer self-assembly was finally performed using PAH and PSS polyelectrolytes to minimize leaching of the biosensor components. The prepared biosensor exhibited linearity over a range of 0.77-2.5mM O-2 (KSV: 0.097/mM of O-2) obtained using from Stern-Volmer plots. The biosensor response to standard cholesterol displayed a linear analytical range from 1.25 to 10mM of cholesterol with regression coefficient of 0.996 (1.25-3.75 mM), 0.976 (1.25-6 mM), and 0.959 (1.25-10mM) and response time of 10 min. Thus, the prepared cholesterol biosensor shows great potential in the diagnosis of hypercholesterolemia. Biotechnol. Bioeng. 2011; 108: 2011-2021. (C) 2011 Wiley Periodicals, Inc.