In our present work, we focused on the terahertz (THz) based characterization of a very efficient organic nonlinear optical (NLO) single crystal of l-alanine (LA). Using the well-known slow evaporation solution growth method (SEST), an optically transparent amino acid-based LA crystal was effectively grown at room temperature. The structural information of the titled crystal was analyzed using powder X-ray diffraction (PXRD) which confirms the space group P212121 and orthorhombic structure of the crystal. Chemical etching of an as-grown single crystal was analyzed to identify the growth patterns. Fourier transform infrared (FTIR) and FT-Raman spectrometry were used to figure out the major functional groups that were present in the ingot crystal. The optical behaviour of LA crystal was observed through UV–Vis and Photoluminescence (PL) spectroscopies. The single crystal was found to have high transmission values over a wide range of wavelength. The PL results revealed that the main emission happens at 474 nm. Thermal stability of the titled compound was acquired by thermo-gravimetric and the differential thermo-gravimetric (TG-DTG) analysis. Open and closed aperture Z-scan measurements were used to analyze the third order nonlinear behavior of the as-grown sample using a femtosecond Ti-sapphire laser. To analyze the absorption peaks behavior Terahertz time-domain spectroscopy (THz-TDS) was also performed in the range 0.2–2.0 THz. The above study shows variation in absorption coefficient and refractive index at THz frequencies.