To provide a scientific basis for simplified, high-efficiency cultivation and precise quality regulation of the elite fresh jujube line MX5N3, this study employed MX5N3 (bred by Tarim University) as the experimental material. During the critical period of fruit development, different concentrations of pyroligneous acid (PA), melatonin (MT), and graphene oxide (GO) were applied via irrigation. Indicators including appearance quality, internal quality, texture properties, and yield were systematically determined. Variance analysis, correlation analysis, and principal component analysis were utilized to analyze fruit quality differences under different biostimulant treatments, and a comprehensive quality evaluation was conducted. Under the evaluation metrics and experimental conditions employed in this study, the treatment with melatonin (0.024‰) yielded the highest comprehensive score. It significantly promoted the accumulation of vitamin C and phenolic substances, enhanced the firmness of both the peel and flesh, and reduced the fruit cracking rate by 57.57%. The GO (0.5‰) treatment increased soluble sugar content and sugar–acid ratio by 14.45% and 27.82%, respectively, yet led to a slight softening of fruit texture. The PA (5‰) treatment increased yield by 62.09% and significantly elevated protein content by 41.48%.