Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single-molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single-molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14 °C. A 10 °C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in neurons.