Abstract Biomimetic scaffolds for tissue engineering should exhibit structural complexity close to native tissues, which typically have non-homogenous nanostructures. This study presents a novel electrospinning process using a cone-shape rotating collector to fabricate polymer nanofiber scaffold with continuous gradient microstructures. The effects of rotation speed (RS) and tip-to-axis distance (TAD) on microstructure gradients were investigated through multivariate analysis of variance (MANOVA), a generalization of univariate ANOVA that allows the researchers to analyze more than one dependent variable in designed experiments. The covariation against multivariate null hypotheses relative to error covariation was visualized through an ellipsoid plot. We found that RS and TAD significantly influenced the gradients in fiber diameter and fiber alignment. The RS was positively correlated to the microstructure gradients, and the TAD was negatively correlated to the gradients. With high TAD, higher RS led to a lower diameter gradient. While with low TAD, higher RS led to higher diameter gradient. In addition, marginal effects on density and mat porosity were also observed. This electrospinning strategy has potential applications in providing biomimetic topological cues for cell matrix with heterogeneous structures.