The effects of dynamic high-pressure microfluidization (DHPM at 400 MPa) and heat treatment (HT) on the microbial inactivation, quality parameters, and flavor components of not-from-concentrate (NFC) cucumber juice were investigated. Total aerobic bacteria, yeasts and molds were not detected in the 400 MPa-treated cucumber juice. Total phenolic content increased by 16.2% in the 400 MPa-treated cucumber juice compared to the control check (CK). The significant reduction in pulp particle size (volume peak decreasing from 100–1000 μm to 10–100 μm) and viscosity increased the stability of the cucumber juice while decreasing the fluid resistance during processing. HT decreased the ascorbic acid content by 25.9% (p < 0.05), while the decrease in ascorbic acid content was not significant after 400 MPa treatment. A total of 59 volatile aroma substances were identified by gas chromatography–ion mobility spectrometry (GC-IMS), and a variety of characteristic aroma substances (i.e., valeraldehyde, (E)-2-hexenal, (E)-2-nonenal, and (E,Z)-2,6-nonadienal, among others) were retained after treatment with 400 MPa. In this study, DHPM technology was innovatively applied to cucumber juice processing with the aim of providing a continuous non-thermal processing technology for the industrial production of cucumber juice. Our results provide a theoretical basis for the application of DHPM technology in cucumber juice production.