DNA barcoding technique is widely used in studying the diversity of copepods and their in situ diet, which promotes the understanding of plankton community structure and marine food web structure. Effective sample preservation is the crucial premise of downstream analysis including PCR amplification and sequencing. In this study, we evaluated the effects of fixatives and storage periods on the yield and quality of the DNA extracted from copepods and their gut content. The integrity of DNA was determined by amplifying and quantifying target fragment of different sizes by qPCR. Flash frozen in liquid nitrogen was used as a control. Considerable variability was observed among different methods. We found that, RNAlater was the best fixative for copepod DNA with highest DNA yield and integrity over two-month storage and ethanol took the second place. For all methods tested, gut content was successfully detected in the DNA extracted from whole organism, but the yield and integrity of prey DNA was independent on that of copepod-specific DNA. Lugol’s and ethanol were the best in preserving the DNA of gut content, indicating the instant stop of copepods’ physiological activity and best permeability. The yield and integrity of copepod DNA progressively decreased with increased storage period for all fixatives, but the storage period did not significantly affected prey DNA. Thus, although obtaining the DNA barcodes from copepods and their gut content both start with whole-organism DNA extraction, the choice of fixative depends on the aim of study.