We have developed ultrasensitive biosensors for the analysis of neurotransmitters such as glutamate, GABA and lactate. These sensors have micrometer to submicrometer sizes. They are based on biomolecule immobilization on optical fiber probe surfaces. The miniaturized fiber probes are fabricated by either pulling or etching conventional optical fibers. For example, surface immobilized glutamate dehydrogenase (GDH) is being used for glutamate analysis. GDH has been directly immobilized onto an optical fiber probe surface through a new optical fiber sensor fabrication technique using covalent binding mechanisms. None of the direct or indirect physical confinement methods, such as mechanical confinement, gel trapping or membrane immobilization, has been used for the sensor preparation. An optical fiber surface is initially activated by silanization, which adds amine groups (-NH2) to the surface. We then affix functional groups -CHO to the optical fiber surface by employing a bifunctional cross-linking agent, glutaraldehyde. The amino acids of GDH enzyme molecules (or other biomolecules) readily attach to these free -CHO groups on the fiber surface. The sensor is able to detect its substrate, glutamate, by monitoring the fluorescence of reduced nicotinamide adenine dinucleotide (NADH), a product of the reaction between nicotinamide adenine dinucleotide (NAD+) and glutamate. Similar procedures and principle have been used for the development of lactate and GABA sensors. Our biomolecule based biosensors have been applied to the study of single living cell neurophysiological responses.