In the past decade, different groups of additives (probiotics, oligosaccharides, and plant extracts) have been widely researched and used in worldwide aquaculture. Recently, other groups, such as biopolymers (e.g., poly‐β‐hydroxybutyrate) with prebiotic properties, have been investigated. Among biopolymers, the use of pure polyhydroxyalkanoate (PHA; composition = 95–100%) as a feed additive in aquaculture has not been studied yet. Therefore, the aim of the present study was to research the effects of four supplementation levels of pure PHA on growth, survival, feed and nutrient utilization, apparent digestibility coefficients (ADCs), and hematology of Nile Tilapia Oreochromis niloticus. Five isonitrogenous and isolipidic diets were designed, including a control (C) diet without PHA supplementation. Remaining diets were supplemented with the following levels of PHA: PHA 0.1% (basal diet + 0.1% PHA), PHA 0.5% (basal diet + 0.5% PHA), PHA 1.0% (basal diet + 1.0% PHA), and PHA 2.0% (basal diet + 2.0% PHA). After a 70‐d feeding test period, the PHA 0.5% and PHA 1.0% experimental groups showed significantly higher final body weight, weight gain, specific growth rate, lipid efficiency and productive value, and ADCs of lipid and energy compared to those of the C group. Protein efficiency ratio was significantly higher in fish fed the PHA 0.1%, PHA 0.5%, and PHA 1.0% diets compared to the C group. Protein productive value significantly increased in the PHA 1.0% group. The ADC of protein was significantly higher in fish fed diets supplemented with any level of PHA compared to C fish. Regarding hematological parameters, hematocrit value showed a significant increase in fish fed PHA 0.1% compared to the C group. Leukocyte composition (%) did not show significant differences among experimental groups. Based on polynomial regression analysis, the optimum inclusion levels of PHA in Nile Tilapia diets were 0.82–0.92% for growth performance, 1.0–1.1% for nutrient utilization, 1.0–1.2% for ADCs, and 1.34% for hematocrit. Cost–benefit ratio analysis revealed nonsignificant but numerical differences among experimental diets. Results suggest that pure PHA, when supplemented at 0.1, 0.5, 1.0, or 2.0%, has certain positive effects on growth, nutrient and feed utilization, and ADCs without compromising Nile Tilapia hematology or health.