Abstract: Stable isotope studies are extremely useful for improving general descriptions of aquatic ecosystems due to their ability to simultaneously summarize complex trophic networks and track the energy flow through them. However, when considering that trophic relationships involve the uncertainty of sample collection, handling and preservation of aquatic organisms, application of general isotopic interpretations may easily lead to misleading conclusions. In these cases, an accepted protocol for the current process in the isotopic research should be considered. In this review, we first measured the uncertainty (isotopic shift) due to sample collection, handling and preservation in a system composed of one consumer and four potential food sources; we then showed four extreme cases to interpret the effect on the relative proportions of sources in the consumer’s diet due to uncertainty in a mixing-polygon model in Isosource. The effects of sample collection, handling and preservation of aquatic organisms on stable isotopes were briefly listed. All these methods had mixed results concerning their effect on the samples tested. For the sample collection, factors, such as, the uniformity, species composition, species life cycles, habitats, tissue difference and turnover, can affect the stable isotope composition. For sample handling, inorganic carbon, gut contents of small animals, and lipid content in the samples were considered as the common factors. For sample preservation, the effects of preservative, time, species or tissue were common on the stable isotope signals. Protocols for sample collection, handling and preservation of aquatic organisms in stable isotope ecology were then recommended. For sample collection, we advise using homogeneous and species-specific samples whenever possible, or performing complementary testing of the bulk- and tissue-specific sampling method. For sample handling, we advocate the gut evacuation for the samples using whole individual, acidification for the particulate organic samples, and lipid extraction and normalization for the samples with high lipid content. For sample preservation, we advocate using fresh material prepared immediately whenever possible, or using drying and freeze-drying when the immediate analysis is not possible, or conducting complementary testing of the preservative method for museum samples. In conclusion, we suggest suitable methods for sample collection, handling and preservation of aquatic organisms for ecological applications of isotopic analysis. The effects of sample collection, handling and preservation on stable isotope ratios need further evaluation. Research is also needed to determine the chemical dynamics in aquatic organisms responsible for the isotopic differences observed among different methods of sample collection, handling and preservation. We encourage further studies of method-specific isotopic variability to improve the normalization for sample collection, handling and preservation of aquatic organisms in stable isotope ecology.