Engineering conducting polymer thin films with morphological homogeneity and long-range molecular ordering is intriguing to achieve high-performance organic electronics. Polyaniline (PANI) has attracted considerable interest due to its appealing electrical conductivity and diverse chemistry. However, the synthesis of large-area PANI thin film and the control of its crystallinity and thickness remain challenging because of the complex intermolecular interactions of aniline oligomers. Here we report a facile route combining air-water interface and surfactant monolayer as templates to synthesize crystalline quasi-two-dimensional (q2D) PANI with lateral size ~50 cm2 and tunable thickness (2.6–30 nm). The achieved q2D PANI exhibits anisotropic charge transport and a lateral conductivity up to 160 S cm−1 doped by hydrogen chloride (HCl). Moreover, the q2D PANI displays superior chemiresistive sensing toward ammonia (30 ppb), and volatile organic compounds (10 ppm). Our work highlights the q2D PANI as promising electroactive materials for thin-film organic electronics.