Two microfluidic devices (pneumatic chip and FlexiChip) have been developed for immobilization and live-intact fluorescence functional imaging of Drosophila larva's Central Nervous System (CNS) in response to controlled acoustic stimulation. The pneumatic chip is suited for automated loading/unloading and potentially allows high throughput operation for studies with a large number of larvae while the FlexiChip provides a simple and quick manual option for animal loading and is suited for smaller studies. Both chips were capable of significantly reducing the endogenous CNS movement while still allowing the study of sound-stimulated CNS activities of Drosophila 3rd instar larvae using genetically encoded calcium indicator GCaMP5. Temporal effects of sound frequency (50-5000 Hz) and intensity (95-115 dB) on CNS activities were investigated and a peak neuronal response of 200 Hz was identified. Our lab-on-chip devices can not only aid further studies of Drosophila larva's auditory responses but can be also adopted for functional imaging of CNS activities in response to other sensory cues. Auditory stimuli and the corresponding response of the CNS can potentially be used as a tool to study the effect of chemicals on the neurophysiology of this model organism.