Auto neuronal synapses, or autapses, are aberrant structures where the synaptic contact of a neuron forms onto its own branch. The functions of autapses, however, remain unknown. Here, we introduce a simple patterning method for capturing a single-cell, in which we maintained the isolated cell until it reached maturity, and developed arrays of autapses for electrophysiological analysis using multi-electrode arrays (MEA). The pattern arrays were formed by selective patterning of poly-L-lysine and various cell repellent materials. We tested the efficiency of single neuron pattern formed according to materials and pattern dimensions. Autapse formation was verified by immunostaining synaptic markers and physiological measurements via recordings from MEA. The results demonstrated that our multiscale patterning method increased the number of autapses consisting of a single neuron, which matured to connect onto themselves. The proposed patterning method (4.06 ± 0.33 isolated single-cells mm-2) is at least twelve times more efficient and productive than the spray method (0.31 ± 0.10 isolated single-cells mm-2). The spontaneous activity of a single neuron on the patterned MEA occured after 11 d in vitro. The single neuron activity consisted of bursts followed by spike trains (the burst rate was 2.56 min-1). This indicates that our method could be used for electrophysiological analysis, including MEA.