Pichia pastoris has been widely used for recombinant protein production on account of its favorable characteristics, especially the tightly regulated alcohol oxidase-1 (AOX-1) promoter. The α-mating factor secretion signal of S. cerevisiae is commonly used for secretion of heterologous proteins in P. pastoris; the efficacy of signal sequence cleavage by Kex2 enzyme in the endoplasmic reticulum may be enhanced by Glu-Ala-containing sequences succeeding the Kex2 cleavage site. In this study, the codon-optimized synthetic gene of A. flavus uricase was cloned in pPICZαA expression vector as two different constructs to examine the role of Glu-Ala-Glu-Ala spacer on enzyme production and secretion. Transformants were screened on agar plates with different concentrations of zeocin (from 200 to 2000 µg/ml) to seek for multi-copy integrants. Among them, the highest enzyme producers with similar uricase encoding gene copy numbers were selected. According to quantitative polymerase chain reaction (qPCR) analysis, the gene copy index was estimated to be 12. Analysis of the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel scan of both constructs showed that the recombinant protein accounted for about 20% of the total secreted protein content in the culture media. Based on enzyme activity measurement, the production yields of 500 and 400 µg/l were achieved for the construct containing the spacer and the construct without the spacer, respectively. It seems that the presence of the spacer does not have any meaningful effect on the expression and secretion of the recombinant uricase in P. pastoris.