Background: The shortnose sturgeon, Acipenser brevirostrum, is an imperiled species distributed along the Atlantic coast of North America. Interest in replenishing wild stocks with hatchery-reared fish has created a need for accurate hematologic and biochemical reference intervals to evaluate the health of both fish raised in aquaculture systems and fish in the wild. Objectives: The objective of this study was to generate hematologic and biochemistry reference intervals for healthy shortnose sturgeon. Methods: Blood samples were collected in heparinized tubes from 77 shortnose sturgeon raised in flow-through aquaculture systems. Whole blood and plasma samples were analyzed for hematologic and biochemical variables using standard techniques. Reference intervals were calculated as the central 95% (percentile) of data. Results: Hematologic reference intervals (n=46) were as follows: PCV 26–46%, hemoglobin 5.7–8.7 g/dL, MCV 307–520 fL, MCH 65.9–107.1 pg, MCHC 15–30 g/dL, plasma proteins (refractometry) 2.8–6.0 g/dL, RBC count 0.65–1.09 × 106/μL, total WBC count 28,376–90,789/μL, small lymphocytes 9063–56,656/μL, large lymphocytes 2122–10,435/μL, neutrophils 3758–33,592/μL, monocytes 0–7137/μL, eosinophils 0–1544/μL, thrombocyte-like cells 6863–23,046/μL, thrombocytes 32,205–122,179/μL, and neutrophil:lymphocyte ratio 0.068–1.026. Plasma chemistry reference intervals (n=77) were as follows: total protein 2.7–5.3 g/dL, albumin 0.8–1.7 g/dL, globulins 1.8–3.7 mg/dL, creatinine 0–1.4 mg/dL, total bilirubin 0–0.1 mg/dL, alkaline phosphatase 47–497 U/L, aspartate aminotransferase 90–311 U/L, sodium 124–141 mmol/L, potassium 2.9–3.7 mmol/L, chloride 106–121 mmol/L, calcium 6.6–12.1 mg/dL, magnesium 1.6–2.3 mg/dL, phosphorus 5.1–8.1 mg/dL, glucose 37–74 mg/dL, cholesterol 42–133 mg/dL, and osmolality 232–289 mOsm/kg. Conclusion: Reference values reported here will be useful for the early detection, identification, and monitoring of disease and sublethal conditions in cultured shortnose sturgeon.