Macroinvertebrates play a key role in aquatic food webs, with amino acids (AA) playing an important role in determining their nutritional value to higher consumers. This study aimed to determine whether AA profile varies among four macroinvertebrate taxa, spatially and seasonally (summer/winter). The freshwater prawn, Macrobrachium australiense; freshwater shrimp, Paratya australiensis; freshwater snail, Physa sp. and caddisfly, Triplectides sp. were collected from seven sites along the Murray–Darling Basin, Australia. Sampling was conducted during summer 2015 (October–December), winter 2016 (May–June) and summer 2016 (October–December). AA profiles were found to be significantly different among the four taxa, with the highest total amino acid content found in decapods (M. australiense and P. australiensis). Based on the total essential AA content from our study, decapods had higher nutritional value compared to Physa. Seasonal variations in AA profiles of decapods were observed, with an increased proportion of the non-essential amino acid glycine and decreased proportion of different essential AAs found in individuals collected during winter compared to summer. No seasonal variation in the AA profiles of Physa sp. or Triplectides sp. was shown. Spatial variation in the AA profile of macroinvertebrates was only recorded in P. australiense during winter 2016. These findings expand the current understanding of the AA profile of freshwater macroinvertebrates showing that AA profiles are taxa specific and vary seasonally and spatially depending on taxa.