The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. Here, using a sensitized triple heterozygote-based genetic screening approach, we report the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. We demonstrate that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, We found that Pyd represses the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, our results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.