Cholesterol is an essential structural lipid of the plasma membrane in eukaryotic cells, which is involved in many physiological functions including the maintenance of the structure of cell membranes and the biosynthesis of various molecules, such as bile acid, vitamin D and testosterone. Its importance does not lay only in its physiological role, but also in its role as a biomarker of several serious diseases (e. g. coronary heart disease, arteriosclerosis and Niemann–Pick disease). Due to its relevance, a great variety of analytical methods have been developed for cholesterol detection in blood, but standard methods suffer from many disadvantages since they are time consuming, labour intensive and require trained personnel. Microfluidics stands as a useful tool to overcome these drawbacks. Within this frame, in this research a new alginate-based biosensor for the colorimetric detection of cholesterol in blood has been developed and integrated into a microfluidic device. By integrating TiO2 nanotubes in the biosensor, cholesterol determination was promoted, lowering the detection time of the assay and improving the image colorimetric analysis of the cholesterol levels. Cholesterol was determined at 7 min with alginate/TiO2 beads, obtaining an LOD and LOQ of 5.0 and 16.7 mM, respectively. Although the assay and its calibration using real blood samples need still to be carried out, the results of this research demonstrate the potential of this platform to become a Point of Care type of device for the fast and reliable determination of cholesterol in blood.