To perform their role effectively, scent detection dogs require certain characteristics. Identifying these characteristics will inform the selection of prospective dogs and preferred approaches to their training. The current study drew upon the perspectives of industry stakeholders to identify the behavioural traits considered relevant for detection dogs in biosecurity screening roles. Dog handlers, trainers, and supervisors (n = 25) in Australian biosecurity operations participated in focus group interviews to determine the perceived characteristics that, in their experience, influence detection performance. Their descriptions were used to create a questionnaire which was then administered to handlers to assess the working behaviours of current biosecurity dogs. Responses were collected for 88% of the operational dogs (n = 36). An exploratory factor analysis revealed seven tentative dimensions: search motivation, emotional stability, search arousal, food motivation, play motivation, search independence, and search focus. Search motivation and search arousal were both positively associated with handler ratings of detection performance (p ≤ 0.006). In general, biosecurity dogs were scored consistently high in ratings of search motivation, emotional stability, and food motivation. Our approach has advanced our understanding of the working behaviours and characteristic profile of biosecurity detector dogs and will be used to inform candidate selection processes.