A new method for generating both continuous and discrete density gradients in microparticles of biodegradable polymers via an electrospray technique is reported. The gradients are generated by spatially varying the deposition time of electrosprayed microparticles. The substrate coated with a density gradient of microparticles has varying surface roughness, offering a unique system for studying the effect of physical cues on neurite outgrowth from dorsal root ganglia. An optimal surface roughness for promoting neuron adhesion and neurite extension in vitro is obtained. Furthermore, this capability of approach is extended to generate a gradient of fluorescein isothiocyanate labeled bovine serum albumin by encapsulating it in the polymer microparticles in situ during electrospray. Taken together, this new class of substrates with gradients of microparticle density can potentially be used in various biomedical applications such as neural tissue engineering.