Increasing urbanization in tropical Africa may create new niches for malaria vectors, potentially leading to higher disease transmission rates. Vector control efforts remain largely targeted at ecologically rural bio-complexities with multiple hosts. Understanding mosquito species composition, ecology, host diversity and biting behavior in urban areas is crucial for planning effective control. This study assessed mosquito species diversity, abundance, behavioral patterns, and Plasmodium sporozoite infection rates of Anopheles vectors along an urban-rural transect in Kisumu city, western Kenya. Indoor and outdoor host-seeking and resting adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and mechanical aspirators (Prokopack) along an urban-rural transect. Females Anopheles mosquitoes collected were identified using morphological taxonomic keys to species level. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further processed using polymerase chain reaction (PCR) to identify members of each complex/group. Subsequently, sporozoite infection rates of the anopheline mosquitoes were determined using a multiplexed real-time quantitative PCR (qPCR) assay. A total of 3,394 female Anopheles mosquitoes were collected and identified. These comprised of An. gambiae s.l. (68%), An. funestus group (19.8%), An. coustani (7.8%), An. pharoensis (2.6%), An. maculipalipis (1.6%), and An. leesoni(0.2%). All six species were found in urban zone, but only three were found in peri-urban and rural sites. Overall, urban collections accounted for the majority of these collections (55.5%) of mosquitoes collected, followed by those from peri-urban (30%) and rural sites (14.5%). Species distribution across the three ecotypes showed Anopheles arabiensis was the dominant species in urban (84.3%) and peri-urban (89%) sites, while An. gambiae s.s. was predominantly found in the rural zone (60.2%) alongside An. arabiensis (39.7%). Anopheles funestus was the predominant species in peri-urban (98.4%) and rural (85.7%) areas, with An. leesoni accounted for 1.6% and 14.3%, respectively. In urban areas, all samples from the An. funestus group were identified as An. funestus s.s.. Majority (55.5%) of Anopheles mosquitoes were collected indoors, while secondary vectors were primarily caught outdoors. Overall, sporozoite rates were higher outdoors 3.5% compared to indoors 1.45% in rural areas. Specifically, sporozoite infectivity rates for An. funestus, An. gambiae s.s and An. arabiensis collected indoors in the rural zone was 2.5%, 1.4% and 1% respectively. Outdoors in rural areas, An. gambiae had a sporozoite rate of 5.3%, while An. arabiensis had a rate of 2.1%. In peri-urban areas An. gambiae had a sporozoite rate of 2.3%. No sporozoites were detected in samples from urban sites. The study highlights a shift of diversity of Anopheles species towards urban areas with increased outdoor activity, and significant outdoor malaria transmission in rural and peri-urban areas, emphasizing the need for tools targeting outdoor-biting mosquitoes. The presence of An. funestus in urban settings is of interest and highlights the critical importance of sustained entomological surveillance to inform integrated vector control and prevent future transmission risks.