The freshwater microcrustacean Dendrocephalus brasiliensis, known as branchoneta, has been considered for use as an alternative live feed for commercial aquatic organisms during larval development. However, there are limited studies on techniques to improve nauplii hatching. Thus, the aim of this work was to develop a method for reproduction and cyst recovery, following a new process for nauplius production by the enzymatic action of chitinase and/or chemical treatment. The physical and chemical water parameters and hatching rate were monitored, while morphological changes on nauplii were evaluated after enzyme and chemical treatment by Scanning Electron Microscopy. A hatching protocol was developed for the use of Ca(OH)2, ascorbic acid and enzyme, wherein the most efficient concentration of enzyme for hatching cysts was determined. The concentrations of commercial chitinase tested were 0.008 U·mL−1, 0.08 U·mL−1 and 0.16 U·mL−1 in 20 mg of cysts, which resulted in the ideal concentration of 0.16 U·mL−1 yielding 88% hatching efficiency. Therefore, the concentration of 0.16 U·mL−1 was used for the second experimental test, which consisted of a control and five experimental treatments with three replicates. In T1, the cysts were subjected to chitinase, T2 to calcium hydroxide, T3 to ascorbic acid, T4 to calcium hydroxide and ascorbic acid and T5 to calcium hydroxide, ascorbic acid and chitinase. The use of the enzyme showed an increase in the hatching rate of the branchoneta, and the addition of calcium hydroxide and ascorbic acid further increased the hatching efficiency of the nauplii (96%). At the naupliar stage one, the treatment with the use of chitinase, calcium hydroxide and ascorbic acid did not present morphological alterations in nauplii. In larval stage two, nauplii already initiate alterations in the region of the abdomen with T5, with segments developed for the naupliar stage. Therefore, the use of enzyme associated with chemicals promotes an increase in hatching when compared to the traditional hatching process. Moreover, this treatment also promotes a reduction of hatching time, as >80% of total hatching was observed on the first day. The proposed protocol could be used for the hatching of branchoneta cysts for the use of nauplii offered as food until naupliar stage 2 in fish and shrimp larviculture. •Chitinase, ascorbic acid and Ca(OH)2 promotes higher hatching efficiency of branchoneta cysts.•The effect of chitinase on nauplii morphogenesis is reduced by the presence of ascorbic acid and Ca(OH)2.•High hatching rates allow large scale production of branchoneta nauplii and biomass.•A new process was stablished, from cyst production and processing to optimizing hatching rates.