The purpose of this study was to investigate the effect of substrate characteristics on the performance of quantum-dot light-emitting diodes (QLEDs) with the aim of developing high-performance flexible QLEDs. Specifically, we compared QLEDs made with a flexible polyethylene naphthalate (PEN) substrate to those made with a rigid glass substrate, using the same materials and structure except for the substrates. Our findings indicate that the PEN QLED had a 3.3 nm wider full width at half maximum and a 6 nm redshifted spectrum in comparison to the glass QLED. Additionally, the PEN QLED exhibited a 6% higher current efficiency, a flatter current efficiency curve, and a 2.25-V lower turn-on voltage, indicating superior overall characteristics. We attribute the spectral difference to the optical properties of the PEN substrate, such as light transmittance and refractive index. Our study also revealed that the electro-optical properties of the QLEDs were consistent with the electron-only device and transient electroluminescence results, which suggests that the improved charge injection properties of the PEN QLED were responsible. Overall, our study provides valuable insights into the relationship between substrate characteristics and QLED performance, which can be used to develop high-performance QLEDs.