A depth-encoding positron emission tomography (PET) detector inserting a horizontal-striped glass between pixilated scintillation crystal layers was developed and experimentally evaluated. The detector consists of 2-layers of 4×4 LYSO array arranged with a 3.37 mm pitch. Horizontal-striped glasses with 1×4 array with different thickness of 3, 4 and 5 mm were inserted between top- and bottom-crystal layers. Bottom surface of bottom-layer was optically coupled to a 4×4 GAPD array. Sixteen output signals from DOI-PET detector were multiplexed by modified resistive charge division (RCD) networks and multiplexed signals were fed into custom-made charge-sensitive preamplifiers. The four amplified signals were digitized and recorded by the custom-made DAQ system based on FPGA. The four digitized outputs were post-processed and converted to flood histograms for each interaction event. Experimental results revealed that all crystal pixels were clearly identified on the 2D flood histogram without overlapping. Patterns of the 2D flood histogram were constituted with arrangements of [bottom–top–bottom–top–\\ldots–top–bottom–top–bottom] crystal responses in X-direction. These could be achieved by employing horizontal-striped glass that controlled the extent of light dispersion towards the X-direction in crystal layers for generation of a different position mapping for each layer and the modified RCD network that controls degree of charge sharing in readout electronics for reduction of identification error. This study demonstrated the proposed DOI-PET detector can extract the 3D γ-ray interaction position without considerable performance degradation of PET detector from the 2D flood histogram.