The present study was conducted to detect the level of serum copper concentration in urban and rural dogs in Ahvaz district, based on the biochemical analysis and atomic absorption spectrophotometery. This survey was accomplished on a group of 250 serum samples of clinically healthy urban and rural dogs with different ages between 2013 and 2014. The urban dogs were selected between referred cases to the Veterinary Hospital of Shahid Chamran University of Ahvaz and the rural dogs were categorized into four areas (north, east, west and south). Classification was made by age, sex, breed, region and diet. The studied dogs were divided into three groups based on age (3 years). The most breed distribution was Mixed (24%), German shepherd (21.6%), Terrier (18.4%), Spitz (11.2%) and Doberman pinscher (9.6%) in the urban dogs. The present results showed that the mean and standard deviation of serum copper concentration was 21.09±0.49 and 15.27±0.29 µmol/L in the urban and rural dogs respectively. Serum copper level was significantly higher in urban dogs than rural dogs (p<0.001) and also in small breeds than large breeds (20.59±4.86 versus 17.27±4.71µmol/L) (p<0.01). The mean of serum copper concentration was significantly higher (p<0.001) in dogs that had meat in their diet (20.64±5.05 versus 12.59±1.18 µmol/L) also. The mean and standard deviation of serum copper were in normal range in all samples (18.10±0.31; 95% CI: 17.16-19.04). Serum copper concentration did not show any significant differences in different age groups, gender and areas (p>0.05). This survey showed that the serum copper concentration was significantly higher in urban dogs than rural dogs and in small breeds than large breeds. It seems that, this study is the first research in dogs in Iran.