The main purpose of this study is to use the experimental and theoretical slow evaporation solution growth technique to examine the structural behavior and NLO activity of a single crystal 4-Picolinium succinate (4PS). Single-crystal XRD investigation revealed the crystallographic system and lattice characteristics, indicating that the produced crystal corresponds to the triclinic system of space group P-1. Theoretically optimized parameters HOMO–LUMO and First-order hyperpolarizability 4PS were also analyzed. The crystal structure was stabilized by N–H…O and O–H…O bond interaction and its super molecular aggregation has been reported. The formed crystal was found to exhibit good crystalline perfection with a full-width at half maximum of 23.4 arc sec, according to high-resolution X-ray diffraction (HRXRD) examinations. Fourier transforms infrared (FTIR) analysis was used to identify the functional groups present in the grown crystal. UV–Visible spectral analysis of the title compound shows a lower cut-off wavelength at 220 nm and optical band gap of 4.7 eV was obtained from Tauc’s plot. Analysis of the 4PS crystal’s fluorescence behavior in the visible spectrum has shown that green color emission occurs at 535 nm. The TG/DTA shows that the studied compound is thermally stable up to 230 °C. The third-order optical nonlinearity of the 4PS crystal has been investigated by using Z-scan method with He–Ne laser pulses at the wavelength of 632 nm. The nonlinear refractive index (n2), nonlinear absorption coefficient (β), and third-order nonlinear susceptibility (χ(3)) are found to be 4.81* 10–19 cm2/W, 3.96* 10–12 cm2/W, and 1.43*10–13 esu, respectively. This signifies that the grown crystal has optical application.