Carbonyl sulfide (OCS) is used to quantify the carbon capture potential of the biosphere because of its direct correlation with CO2 uptake during photosynthesis. However, to constrain the urban biosphere signal, it is necessary to evaluate potential anthropogenic sources. We conducted two sampling campaigns in the Metropolitan Area of Barcelona (AMB), Spain, during May (full COVID lockdown) and October 2020 to measure the spatial distribution and variability of OCS in four urban land uses as follows: built, urban forest, urban park, and peri-urban agriculture. The OCS background levels determined at Tibidabo (442 m asl) were approximately 484 ±20 ppt and 407 ±8 ppt for May and October 2020, respectively, and agreed with other seasonal surveys conducted in Europe during that same period. The urban values ranged from neutral to above background, suggesting nearby anthropogenic and marine emissions such as +D150 ppt in Montjuic, which is downwind of Barcelona’s harbor. During the crop-growing season in May, the agricultural areas consistently showed values below the background (uptake) at 7:00 UTC when the land breezes were dominant, while later in the morning, when the sea breeze are developed, the plant sink is masked by the transport of marine emissions. Urban forests located north of Tibidabo showed OCS values up to -D70 ppt, suggesting significant uptake by urban forests. We conclude that determining the urban biosphere signal using OCS as a tracer is more complex than expected because the marine and anthropogenic emissions from the port strongly impact the spatial-temporal distribution of OCS.