Fruit development is influenced greatly by endogenous hormones including salicylic acid (SA) and abscisic acid (ABA). Mass spectrometry with high sensitivity has become a routine technology to analyze hormones. However, pretreatment of plant samples remains a difficult problem. Thus, dispersive liquid–liquid microextraction (DLLME) was used to concentrate trace plant hormones before liquid chromatography–ion trap mass spectrometry (LC–ITMS) analysis. Standard curves were linear within the ranges of 0.5–50, 0.2–20 ng/mL for SA and ABA, respectively. The correlation coefficients were greater than 0.9995 with recoveries above 87.5%. The limits of detection were 0.2 ng/mL for SA and 0.1 ng/mL for ABA in spiked water solution, respectively (injection 20 μL). The successful analysis of SA and ABA in fruit samples indicated our DLLME–LC–ITMS approach was efficient, allowing reliable quantification of both two compounds from very small amounts of plant material. Moreover, this research revealed the relationship between SA and ABA content and development of peach fruit at different growth stages.