Amperometric biosensors are complex systems and they require a combination of technologies for their development. The aim of the present work is to exploit existing technologies for new purposes in order to develop nanostructured biosensors for the real-time detection of multiple metabolites in cell culture flasks. The fabrication of five Au working electrodes onto silicon substrate is achieved with CMOS compatible microtechnology. Each working electrode presents an area of 0.25 mm², so structuration with carbon nanotubes and specific functionalization are carried out by using spotting technology, originally developed for microarrays and DNA printing. The electrodes are characterized by cyclic voltammetry and compared with commercially available screen-printed electrodes. Finally, they are functionalized with different enzymes and calibrated for real-time detection of glutamate, glucose and lactate under flow conditions. The developed biosensor for real-time and online detection of multiple metabolites shows very promising results towards its application in cell culture monitoring.