Chitosan nanoparticles (ChNPs) as a biodegradable and biocompatible substance were employed as a bio-elicitor to stimulate the biosynthesis of bioactive metabolites in avocado callus. Effect of in-situ encapsulation of different active compounds (Salicylic acid, Phenylalanine, Methionine and Ca-Pyruvate) onto ChNPs during preparation was performed using ionic gelation method. The prepared encapsulated nanoparticles were characterized using transmission electron microscope (TEM) and dynamic light scattering (DLS) techniques. The formulated ChNPs had a beneficial effect on the majority of the indicators tested on Murashige and Skoog (MS) medium. The results indicated that maximum mean value of callus fresh weight before the addition of nano-Chitosan was 215 mg/jar, and ChNPs encapsulated methionine 40 mg/L had the greatest mean record of callus fresh weight and glutathione content with low malondialdehyde (MDA) concentration of 0.993 nmol/g. However, ChNPs encapsulated salicylic acid at 100mg/L has the best antioxidant capacity due to a 40.88% rise in the content of the essential oil 3,4-Dihydro-2H-1,5-(3"-T-butyl) benzodioxepine and the largest accumulation of polyphenolic components. The current approach involved analysing molecular amplified fragment-length polymorphism (AFLP) using genomic DNA with two restriction enzymes (EcoRI and MseI) EcoR I-ACA and MseI-CTC primer pairs. The present work provides an efficient method for enhancing the bio-active compounds bioaccumulation in avocado callus using encapsulated ChNPs.