Fish are the most numerous pet throughout the United States, and ornamental Koi ( Cyprinus rubrofuscus ) are one of the most common ornamental pet fish in California, USA. Previous reference intervals have been established in a small sample size of koi but differences between age and sex in ornamental koi have not been investigated. The objective of this study was to establish hematologic and biochemical reference intervals for healthy koi stratified by age (juvenile vs adult) and sex (male vs female). Blood samples were collected from 50 adult males, 50 adult females and 50 juvenile koi from a commercial vendor. Heparinized whole blood was used to perform a manual complete blood count (CBC) and biochemical parameters were analyzed from plasma samples. Reference intervals were determined using a non-parametric method to obtain the 2.5% and 97.5% percentiles following elimination of outliers. Many of the investigated laboratory values were found to be similar between groups, but statistically significant differences were found between ages for calcium, anion gap, and thrombocyte count, and among both age and sex for phosphorus, bicarbonate, aspartate aminotransferase (AST), glucose, red blood cell count (RBC), white blood cell count (WBC), and lymphocyte count. Statistically significant differences between ages and sexes were found to exist for various analytes, but the differences are unlikely to be clinically relevant given the large degree of overlap of the partitioned reference intervals. Overall, reference intervals for the investigated hematological and biochemical values were similar in nonbreeding koi regardless of sex and age.