Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.