Living cells can change their intramembranous temperature during cell activities such as division, gene expression, enzyme reaction, and metabolism [1,2]. Moreover, under external stimuli, such as drugs or other signals, cells may quickly change their metabolic activities, leading to acute variation of intracellular temperatures from the normal state [3,4]. However, such temperature change inside cells is usually at a small scale and is of transient nature due to the thermo-influence by the extracellular environment, rendering it rather difficult to measure using the conventional temperature detection methods. Thus, a more precise and faster-response thermometer is needed to measure single-cell temperature changes in real time, which may constitute a new layer of cellular information for studies of cellular signaling, and even clinical diagnosis and therapy.Fluorescent nanogel has been previously applied to detect changes in intracellular temperature [4]. Cells were first allowed to take up a fluorescence material and the average intracellular temperature change under a certain treatment was then determined through measuring the distinct fluorescent light intensity before and after the treatment. Such a fluorescent nanogel-based method has a number of disadvantages, including potential toxicity to cells, limit of measurement resolution (generally in the range of 0.29 °C-0.50 °C), and limit of time-scale resolution (at the scale of minutes).Thermocouple (TC) is widely used in settings that require detection of temperature changes. The TC-based detection method has a number of advantages, including the capacity for achieving high precision and rapid response. To adapt the TC method for temperature measurement at the single-cell level, one would need to develop a micro-sized TC probe (at sub-micrometer scale). The thin film method is a common approach to producing two-dimensional micro-or even nano-TCs for use in electronics industry [5]. However, such two-dimensional TCs that rely on the support of silicon chips cannot be readily used for measuring intracellular temperature. In this report, we designed a novel TC device for detecting intracellular temperature ( Figure 1A and 1B). Briefly, our TC probe is made of a sandwich structure consisting of the tungsten (W) substrate, an insulating layer made of polyurethane (PU; except at the tip), and a platinum (Pt) film (Supplementary information, Figure S1). We produced two types of TC probes, with different thickness of the Pt film (50 nm and 100 nm). In a calibration experiment with these two types of probes, we found that the 100 nm probe produced a temperature-thermoelectricity curve that showed an almost perfect match with the standard curve produced by a regular macro-sized TC, while the readings from the 50 nm probe showed deviations from the standard curve ( Figure 1B and 1C and Supplementary information, Figure S2). This result is consistent with earlier reports that when the thickness of the Pt film decreases beyond the 100 nm range, it will affect the resulti...