Leptospirosis is a bacterial disease caused by pathogenic Leptospira that infect both human and animals. This study was conducted to determine the seroprevalence of leptospirosis among rangers and wild animals in two ex-situ captive centers, Bukit Merah Orangutan Island (BMOUI) and Taiping Zoo, Perak and to identify the risk factors responsible for the leptospiral seropositivity. Blood samples from rangers and animals of BMOUI and Taiping Zoo were taken to determine the presence of antibodies against Leptospira through microscopic agglutination test (MAT) using 21 serovars of Leptospira commonly found in Malaysia as antigens. Structured surveys in identifying risk factors were given to each ranger from both study sites. It was observed that Rattus exulans (1/10) (10.0%), Rattus rattus (1/5) (20.0%), Niniventer fulvescens (1/1) (100.0%), Callosciurus notatus (0/6) (0.0%), Tupaia tana (1/1) (100.0%), Pongo pygmaeus (5/10) (50.0%) and BMOUI rangers (8/18) (44.4%) were positive for leptospiral antibodies. Samples obtained from Taiping Zoo also revealed the presence of leptospiral antibodies in R. rattus (0/19) (0.0%), R. exulans (1/2) (50.0%) and rangers (2/5) (40.0%). Among the positive cases, most human and animal samples from both study sites reacted with serovar Lepto 175. Our surveys indicated no significant associations between seroprevalence of leptospiral antibodies with rangers’ age (p = 0.82), sex (p = 0.85), ethnicity (p = 0.65), educational level (p = 0.88) and working experience (p = 0.82). In terms of risk factors, no significant associations between seroprevalence of leptospiral antibodies with knowledge on leptospirosis (p = 0.82), working hours (p = 0.53), smoking (p = 0.85), crossing rivers/pools/stagnant water while working (p = 0.90) and wearing full personal protective equipment (PPE) (p = 0.73). This study provides epidemiological data on leptospirosis in rangers and animals from BMOUI and Taiping Zoo which is of paramount importance for improving strategies in prevention of the disease.