Since alcohol and tobacco consumption are among the leading causes of population health harm, it is very important to understand the consumption behaviour to develop effective harm reduction strategies. Wastewater-based epidemiology (WBE) is a potential tool for estimating their consumption, but there are several uncertainties that need to be determined, including the stability of biomarkers in the sewer. Utilizing a real rising main sewer, this study investigated the stability of alcohol and tobacco consumption biomarkers. Rhodamine and acesulfame were used as flow tracer and benchmarker to understand the transportation of wastewater in the sewer with a hydraulic retention time between 2.7 and 5.0 h. Ethyl sulphate (EtS) and ethyl glucuronide (EtG), two biomarkers of alcohol consumption, were found to have different in-sewer stability, with EtS much more stable than EtG. The degradation rate of EtS is approximately 8% per hour, while EtG has a half-life of 1.9 h. Formation of nicotine, cotinine and trans-3′-hydroxycotinine, three biomarkers for tobacco consumption, was observed during the experiment, probably due to deconjugation of their glucuronide chemicals. The deconjugation process has prevented the determination of actual stability of the three chemicals. However, it is suggested that cotinine is relatively stable, while nicotine and trans-3′-hydroxycotinine degrade to a certain degree in the sewer system. According to our findings, the in-sewer degradation is more important during the interpretation of alcohol consumption estimation than for tobacco consumption estimation.