Medical disorders caused by second-hand smoke are a major public health concern worldwide. To estimate the level of second-hand smoke exposure, salivary diagnostics for cotinine analysis is a compelling alternative in conventional diagnostics using bio-fluids, such as blood and urine, owing to its simple and non-invasive collection method. However, there are several critical issues, such as tedious multisteps, demand for expertise, and field unavailability to collect and transport the purified saliva for further analysis. Here, an all-in-one platform is presented to simply collect real human saliva and directly deliver it onto the biosensing surface. The platform consists of a commercial cotton-swab-type collector, 3D-printed housing, and microfluidic channel integrated with an electrochemical competitive immunosensor to evaluate the level of salivary cotinine. The immunosensor is based on a competitive binding assay between cotinine-conjugated horseradish peroxidase (C-HRP) and cotinine for anti-cotinine binding sites. The current responses obtained from the HRP-thionine-H2O2 system decreased proportionally to the cotinine concentration. This immunosensor successfully detected its target over a range of 1 × 10-1 to 1 × 104 pg ml-1 with a low limit of detection of 6 × 10-2 pg ml-1 and a limit of quantification of 1 × 10-1 pg ml-1. In addition, the platform is applicable to various commercial cotton-swab-type saliva collectors and can successfully transfer the saliva in wide flow rates ranging from 0.1 to 30 ml min-1 without leakage or damage to the sensing surface. Furthermore, the practicality of the proposed platform was evaluated by measuring cotinine in real human saliva from eight non-smokers. The concentration of cotinine was from 45.7 to 890.8 pg ml-1, which was in good agreement with that measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The introduced all-in-one platform represented a reliable performance delivering simple and practical steps in salivary diagnostics.