Uniform alignment of rigid-rod liquid crystal (LC) molecules under applied voltage is critical for achievement of high-quality display for thin-film transistor-driven liquid crystal display devices (TFT-LCDs). The polymeric components that can induce the alignment of randomly aligned LC molecules are called alignment layers (ALs). In the current work, a series of organo-soluble polyimide (SPI) ALs were designed and prepared from an alicyclic dianhydride, hydrogenated 3,3′,4,4′-biphenyltetracarboxylic dianhydride (HBPDA), and various aromatic diamines, including 4,4′-methylenedianiline (MDA) for SPI-1, 4,4′-aminodianiline (NDA) for SPI-2, 3,3′,5,5′-tetramethyl-4,4′-diaminodiphenylmethane (TMMDA) for SPI-3, and 3,3′-diethyl-5,5′-dimethyl-4,4′-diaminodiphenylmethane (DMDEDA) for SPI-4. The derived SPI resins were all soluble in N-methyl-2-pyrrolidone (NMP). Four SPI alignment agents with the solid content of 6 wt.% were prepared by dissolving the SPI resins in the mixed solvent of NMP and butyl cellulose (BC) (NMP/BC = 80:20, weight ratio). Liquid crystal minicells were successfully fabricated using the developed SPI varnishes as the LC molecule alignment components. The SPI ALs showed good alignment ability for the LC molecules with the pretilt angles in the range of 1.58°–1.97°. The LC minicells exhibited good optoelectronic characteristics with voltage holding ratio (VHR) values higher than 96%. The good alignment ability of the SPI ALs is mainly attributed to the good comprehensive properties of the SPI layers, including high volume resistivity, high degree of imidization at the processing temperature (230 °C), good rubbing resistance, good thermal stability with glass transition temperatures (Tgs) higher than 260 °C, and excellent optical transparency with the transmittance higher than 97% at the wavelength of 550 nm.